Eisenberg, David S.


Eisenberg David


David Eisenberg is currently Professor of Chemistry and Biochemistry and Biological Chemistry, as well as HHMI Investigator and Director of the UCLA-DOE Institute for Genomics and Proteomics. Before he came to UCLA, Eisenberg earned an A.B. in Biochemical Sciences from Harvard College and a D.Phil. from Oxford University in Theoretical Chemistry on a Rhodes Scholarship. After postdoctoral study at Princeton University on water and hydrogen bonding and at Caltech on protein crystallography, he joined the faculty at UCLA. Currently he studies protein interactions by X-ray crystallography, bioinformatics, and biochemistry, with an emphasis on amyloid-forming proteins. This recently recognized protein state offers opportunities to understand cells in health and disease, and in synthesizing new materials and in understanding processes as diverse as biofilms and corrosion. Eisenberg has published over 300 papers and reviews, and holds half a dozen patents. His awards include: the UCLA Distinguished Teaching Award, John Simon Guggenheim Fellowship, the UCLA Faculty Research Lectureship, the Stein and Moore Award of the Protein Society, the ACS Faculty Mentoring Award, and membership in the National Academy of Sciences, the American Academy of Arts and Sciences, the American Philosophical Society, and the Institute of Medicine.

Research Interests

David Eisenberg and his research group focus on protein interactions. In their experiments they study the structural basis for conversion of normal proteins to the amyloid state and conversion of prions to the infectious state. In bioinformatic work, they derive information on protein interactions from genomic and proteomic data, and design inhibitors of amyloid toxicity.

Amyloid and prion diseases are diseases of protein aggregation in which a normal, functional protein converts to an abnormal, aggregated protein. The systemic amyloid diseases, such as dialysis-related amyloidosis, are apparently caused by the accumulation of fibers until organs fail. The neurodegenerative amyloid diseases, such as Alzheimer’s, Parkinson’s, amyotrophic lateral sclerosis (ALS), and the prion conditions, seem to be caused by smaller oligomers, intermediate in size between monomers and fibers. Our goals are to understand the general features of the conversion to the amyloid state, why some of the diseases are transmissible between organisms and others not, what the structures of the toxic units are, and how they exert their toxic actions.

In 2005, we determined the atomic-level structure for the spine of an amyloid fiber. This structure shows that the spine consists of two parallel beta sheets, packed across a tight, dry interface that we call a steric zipper. The structure of the spine explains the stability of amyloid, gives hints about the conversion process, and suggests why some proteins form amyloid while others do not. Since 2005, we have determined some 90 amyloid spines from 15 disease-related proteins, using a combination of bioinformatics and structural tools. In 2010, we determined the structure of a small toxic amyloid-related oligomer, consisting of six anti-parallel beta strands forming a cylindrical barrel. This structure may suggest models for the toxic oligomers associated with amyloid diseases.

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Honors & Awards

  • UCLA McCoy Award
  • Amgen Award of the Protein Society
  • Pierce Award of the Immunotoxin Society
  • Biophysical Society Emily M. Gray Award
  • Technion – Israel Institute of Technology Harvey Prize in Human Health
  • Preceptor for the 2009 Nobel Laureate Signature Award for Graduate Education in Chemistry
  • Harvard Westheimer Medal
  • UCLA Seaborg Medal
  • Howard Hughes Medical Institute Investigator
  • National Academy of Sciences Elected Member
  • American Association for the Advancement of Science Fellow
  • American Philosophical Society Member
  • Institute of Medicine Member

Representative Publications

Below is the list of the most recent and selected publications.  For the full list, please visit here.








  • Khakshoor O, Lin AJ, Korman TP, Sawaya MR, Tsai SC, Eisenberg D, Nowick JS. (2010). X-ray crystallographic structure of an artificial beta-sheet dimer. J Am Chem Soc.. Aug 2010. 25;132(33): 11622-8 [Abstract]

    This paper describes the X-ray crystallographic structure of a designed cyclic beta-sheet peptide that forms a well-defined hydrogen-bonded dimer that mimics beta-sheet dimers formed by proteins. The 54-membered ring macrocyclic peptide (1a) contains molecular template and turn units that induce beta-sheet structure in a heptapeptide strand that forms the dimerization interface. The X-ray crystallographic structure reveals the structures of the two “Hao” amino acids that help template the beta-sheet structure and the two delta-linked ornithine turn units that link the Hao-containing template to the heptapeptide beta-strand. The Hao amino acids adopt a conformation that resembles a tripeptide in a beta-strand conformation, with one edge of the Hao unit presenting an alternating array of hydrogen-bond donor and acceptor groups in the same pattern as that of a tripeptide beta-strand. The delta-linked ornithines adopt a conformation that resembles a hydrogen-bonded beta-turn, in which the ornithine takes the place of the i+1 and i+2 residues. The dimers formed by macrocyclic beta-sheet 1a resemble the dimers of many proteins, such as defensin HNP-3, the lambda-Cro repressor, interleukin 8, and the ribonuclease H domain of HIV-1 reverse transcriptase. The dimers of 1a self-assemble in the solid state into a barrel-shaped trimer of dimers in which the three dimers are arranged in a triangular fashion. Molecular modeling in which one of the three dimers is removed and the remaining two dimers are aligned face-to-face provides a model of the dimers of dimers of closely related macrocyclic beta-sheet peptides that were observed in solution.

  • Goldschmidt L, Teng PK, Riek R, Eisenberg D. (2010). Identifying the amylome, proteins capable of forming amyloid-like fibrils.Proc. Natl. Acad. Sci. U.S.A.. Feb 2010. 107(8):3487-92. [Abstract]

    The amylome is the universe of proteins that are capable of forming amyloid-like fibrils. Here we investigate the factors that enable a protein to belong to the amylome. A major factor is the presence in the protein of a segment that can form a tightly complementary interface with an identical segment, which permits the formation of a steric zipper-two self-complementary beta sheets that form the spine of an amyloid fibril. Another factor is sufficient conformational freedom of the self-complementary segment to interact with other molecules. Using RNase A as a model system, we validate our fibrillogenic predictions by the 3D profile method based on the crystal structure of NNQQNY and demonstrate that a specific residue order is required for fiber formation. Our genome-wide analysis revealed that self-complementary segments are found in almost all proteins, yet not all proteins form amyloids. The implication is that chaperoning effects have evolved to constrain self-complementary segments from interaction with each other.

  • Laganowsky A, Benesch JL, Landau M, Ding L, Sawaya MR, Cascio D, Huang Q, Robinson CV, Horwitz J, Eisenberg D. (2010). Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function.Protein Sci.. May 2010. 19(5):1031-43. [Abstract]

    Small heat shock proteins alphaA and alphaB crystallin form highly polydisperse oligomers that frustrate protein aggregation, crystallization, and amyloid formation. Here, we present the crystal structures of truncated forms of bovine alphaA crystallin (AAC(59-163)) and human alphaB crystallin (ABC(68-162)), both containing the C-terminal extension that functions in chaperone action and oligomeric assembly. In both structures, the C-terminal extensions swap into neighboring molecules, creating runaway domain swaps. This interface, termed DS, enables crystallin polydispersity because the C-terminal extension is palindromic and thereby allows the formation of equivalent residue interactions in both directions. That is, we observe that the extension binds in opposite directions at the DS interfaces of AAC(59-163) and ABC(68-162). A second dimeric interface, termed AP, also enables polydispersity by forming an antiparallel beta sheet with three distinct registration shifts. These two polymorphic interfaces enforce polydispersity of alpha crystallin. This evolved polydispersity suggests molecular mechanisms for chaperone action and for prevention of crystallization, both necessary for transparency of eye lenses.

  • Wang L, Schubert D, Sawaya MR, Eisenberg D, Riek R. (2010). Multidimensional structure-activity relationship of a protein in its aggregated states.Angew. Chem. Int. Ed. Engl.. May 2010. 49(23):3904-8. [Abstract]
  • Khakshoor O, Lin AJ, Korman TP, Sawaya MR, Tsai SC, Eisenberg D, Nowick JS. (2010). X-ray crystallographic structure of an artificial beta-sheet dimer.J. Am. Chem. Soc.. Aug 2010. 132(33):11622-8. [Abstract]

    This paper describes the X-ray crystallographic structure of a designed cyclic beta-sheet peptide that forms a well-defined hydrogen-bonded dimer that mimics beta-sheet dimers formed by proteins. The 54-membered ring macrocyclic peptide (1a) contains molecular template and turn units that induce beta-sheet structure in a heptapeptide strand that forms the dimerization interface. The X-ray crystallographic structure reveals the structures of the two “Hao” amino acids that help template the beta-sheet structure and the two delta-linked ornithine turn units that link the Hao-containing template to the heptapeptide beta-strand. The Hao amino acids adopt a conformation that resembles a tripeptide in a beta-strand conformation, with one edge of the Hao unit presenting an alternating array of hydrogen-bond donor and acceptor groups in the same pattern as that of a tripeptide beta-strand. The delta-linked ornithines adopt a conformation that resembles a hydrogen-bonded beta-turn, in which the ornithine takes the place of the i+1 and i+2 residues. The dimers formed by macrocyclic beta-sheet 1a resemble the dimers of many proteins, such as defensin HNP-3, the lambda-Cro repressor, interleukin 8, and the ribonuclease H domain of HIV-1 reverse transcriptase. The dimers of 1a self-assemble in the solid state into a barrel-shaped trimer of dimers in which the three dimers are arranged in a triangular fashion. Molecular modeling in which one of the three dimers is removed and the remaining two dimers are aligned face-to-face provides a model of the dimers of dimers of closely related macrocyclic beta-sheet peptides that were observed in solution.

  • Apostol MI, Sawaya MR, Cascio D, Eisenberg D. (2010). Crystallographic studies of prion protein (PrP) segments suggest how structural changes encoded by polymorphism at residue 129 modulate susceptibility to human prion disease.J. Biol. Chem.. Sep 2010. 285(39):29671-5. [Abstract]

    A single nucleotide polymorphism (SNP) in codon 129 of the human prion gene, leading to a change from methionine to valine at residue 129 of prion protein (PrP), has been shown to be a determinant in the susceptibility to prion disease. However, the molecular basis of this effect remains unexplained. In the current study, we determined crystal structures of prion segments having either Met or Val at residue 129. These 6-residue segments of PrP centered on residue 129 are “steric zippers,” pairs of interacting β-sheets. Both structures of these “homozygous steric zippers” reveal direct intermolecular interactions between Met or Val in one sheet and the identical residue in the mating sheet. These two structures, plus a structure-based model of the heterozygous Met-Val steric zipper, suggest an explanation for the previously observed effects of this locus on prion disease susceptibility and progression.

  • Arbing MA, Kaufmann M, Phan T, Chan S, Cascio D, Eisenberg D. (2010). The crystal structure of the Mycobacterium tuberculosis Rv3019c-Rv3020c ESX complex reveals a domain-swapped heterotetramer.Protein Sci.. Sep 2010. 19(9):1692-703. [Abstract]

    Mycobacterium tuberculosis encodes five gene clusters (ESX-1 to ESX-5) for Type VII protein secretion systems that are implicated in mycobacterial pathogenicity. Substrates for the secretion apparatus are encoded within the gene clusters and in additional loci that lack the components of the secretion apparatus. The best characterized substrates are the ESX complexes, 1:1 heterodimers of ESAT-6 and CFP-10, the prototypical member that has been shown to be essential for Mycobacterium tuberculosis pathogenesis. We have determined the structure of EsxRS, a homolog of EsxGH of the ESX-3 gene cluster, at 1.91 Å resolution. The EsxRS structure is composed of two four-helix bundles resulting from the 3D domain swapping of the C-terminal domain of EsxS, the CFP-10 homolog. The four-helix bundles at the extremities of the complex have a similar architecture to the structure of ESAT-6·CFP-10 (EsxAB) of ESX-1, but in EsxRS a hinge loop linking the α-helical domains of EsxS undergoes a loop-to-helix transition that creates the domain swapped EsxRS tetramer. Based on the atomic structure of EsxRS and existing biochemical data on ESX complexes, we propose that higher order ESX oligomers may increase avidity of ESX binding to host receptor molecules or, alternatively, the conformational change that creates the domain swapped structure may be the basis of ESX complex dissociation that would free ESAT-6 to exert a cytotoxic effect.

  • Laganowsky A, Eisenberg D. (2010). Non-3D domain swapped crystal structure of truncated zebrafish alphaA crystallin.Protein Sci.. Oct 2010. 19(10):1978-84. [Abstract]

    In previous work on truncated alpha crystallins (Laganowsky et al., Protein Sci 2010; 19:1031-1043), we determined crystal structures of the alpha crystallin core, a seven beta-stranded immunoglobulin-like domain, with its conserved C-terminal extension. These extensions swap into neighboring cores forming oligomeric assemblies. The extension is palindromic in sequence, binding in either of two directions. Here, we report the crystal structure of a truncated alphaA crystallin (AAC) from zebrafish (Danio rerio) revealing C-terminal extensions in a non three-dimensional (3D) domain swapped, “closed” state. The extension is quasi-palindromic, bound within its own zebrafish core domain, lying in the opposite direction to that of bovine AAC, which is bound within an adjacent core domain (Laganowsky et al., Protein Sci 2010; 19:1031-1043). Our findings establish that the C-terminal extension of alpha crystallin proteins can be either 3D domain swapped or non-3D domain swapped. This duality provides another molecular mechanism for alpha crystallin proteins to maintain the polydispersity that is crucial for eye lens transparency.


  • Sprinzak E, Cokus SJ, Yeates TO, Eisenberg D, Pellegrini M. (2009). Detecting coordinated regulation of multi-protein complexes using logic analysis of gene expression.BMC Syst Biol. 2009. 3:115. [Abstract]

    Many of the functional units in cells are multi-protein complexes such as RNA polymerase, the ribosome, and the proteasome. For such units to work together, one might expect a high level of regulation to enable co-appearance or repression of sets of complexes at the required time. However, this type of coordinated regulation between whole complexes is difficult to detect by existing methods for analyzing mRNA co-expression. We propose a new methodology that is able to detect such higher order relationships.

  • Miallau L, Faller M, Chiang J, Arbing M, Guo F, Cascio D, Eisenberg D. (2009). Structure and proposed activity of a member of the VapBC family of toxin-antitoxin systems. VapBC-5 from Mycobacterium tuberculosis.J. Biol. Chem.. Jan 2009. 284(1):276-83. [Abstract]

    In prokaryotes, cognate toxin-antitoxin pairs have long been known, but no three-dimensional structure has been available for any given complex from Mycobacterium tuberculosis. Here we report the crystal structure and activity of a member of the VapBC family of complexes from M. tuberculosis. The toxin VapC-5 is a compact, 150 residues, two domain alpha/beta protein. Bent around the toxin is the VapB-5 antitoxin, a 33-residue alpha-helix. Assays suggest that the toxin is an Mg-enabled endoribonuclease, inhibited by the antitoxin. The lack of DNase activity is consistent with earlier suggestions that the complex represses its own operon. Furthermore, analysis of the interactions in the binding of the antitoxin to the toxin suggest that exquisite control is required to protect the bacteria cell from toxic VapC-5.

  • Wiltzius JJ, Sievers SA, Sawaya MR, Eisenberg D. (2009). Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process.Protein Sci.. Jul 2009. 18(7):1521-30. [Abstract]

    Islet Amyloid Polypeptide (IAPP or amylin) is a peptide hormone produced and stored in the beta-islet cells of the pancreas along with insulin. IAPP readily forms amyloid fibrils in vitro, and the deposition of fibrillar IAPP has been correlated with the pathology of type II diabetes. The mechanism of the conversion that IAPP undergoes from soluble to fibrillar forms has been unclear. By chaperoning IAPP through fusion to maltose binding protein, we find that IAPP can adopt a alpha-helical structure at residues 8-18 and 22-27 and that molecules of IAPP dimerize. Mutational analysis suggests that this dimerization is on the pathway to fibrillation. The structure suggests how IAPP may heterodimerize with insulin, which we confirmed by protein crosslinking. Taken together, these experiments suggest the helical dimerization of IAPP accelerates fibril formation and that insulin impedes fibrillation by blocking the IAPP dimerization interface.

  • Teng PK, Eisenberg D. (2009). Short protein segments can drive a non-fibrillizing protein into the amyloid state.Protein Eng. Des. Sel.. Aug 2009. 22(8):531-6. [Abstract]

    Protein fibrils termed amyloid-like are associated with numerous degenerative diseases as well as some normal cellular functions. Specific short segments of amyloid-forming proteins have been shown to form fibrils themselves. However, it has not been shown in general that these segments are capable of driving a protein from its native structure into the amyloid state. We applied the 3D profile method to identify fibril-forming segments within the amyloid-forming human proteins tau, alpha-synuclein, PrP prion and amyloid-beta. Ten segments, six to eight residues in length, were chosen and inserted into the C-terminal hinge loop of the highly constrained enzyme RNase A, and tested for fibril growth and Congo red birefringence. We find that all 10 unique inserts cause RNase A to form amyloid-like fibrils which display characteristic yellow to apple-green Congo red birefringence when observed with cross polarizers. These six to eight residue inserts can fibrillize RNase A and are sufficient for amyloid fibril spine formation.

  • Wiltzius JJ, Landau M, Nelson R, Sawaya MR, Apostol MI, Goldschmidt L, Soriaga AB, Cascio D, Rajashankar K, Eisenberg D. (2009). Molecular mechanisms for protein-encoded inheritance.Nat. Struct. Mol. Biol.. Sep 2009. 16(9):973-8. [Abstract]

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein ‘conformations’. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of beta-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct beta-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  • Ivanova MI, Sievers SA, Sawaya MR, Wall JS, Eisenberg D. (2009). Molecular basis for insulin fibril assembly.Proc. Natl. Acad. Sci. U.S.A.. Nov 2009. 106(45):18990-5. [Abstract]

    In the rare medical condition termed injection amyloidosis, extracellular fibrils of insulin are observed. We found that the segment of the insulin B-chain with sequence LVEALYL is the smallest segment that both nucleates and inhibits the fibrillation of full-length insulin in a molar ratio-dependent manner, suggesting that this segment is central to the cross-beta spine of the insulin fibril. In isolation from the rest of the protein, LVEALYL forms microcrystalline aggregates with fibrillar morphology, the structure of which we determined to 1 A resolution. The LVEALYL segments are stacked into pairs of tightly interdigitated beta-sheets, each pair displaying the dry steric zipper interface typical of amyloid-like fibrils. This structure leads to a model for fibrils of human insulin consistent with electron microscopic, x-ray fiber diffraction, and biochemical studies.

  • Salwinski L, Licata L, Winter A, Thorneycroft D, Khadake J, Ceol A, Aryamontri AC, Oughtred R, Livstone M, Boucher L, Botstein D, Dolinski K, Berardini T, Huala E, Tyers M, Eisenberg D, Cesareni G, Hermjakob H. (2009). Recurated protein interaction datasets.Nat. Methods. Dec 2009. 6(12):860-1. [Abstract]


  • Riley R, Pellegrini M, Eisenberg D. (2008). Identifying cognate binding pairs among a large set of paralogs: the case of PE/PPE proteins of Mycobacterium tuberculosis.PLoS Comput. Biol.. 2008. 4(9):e1000174. [Abstract]

    We consider the problem of how to detect cognate pairs of proteins that bind when each belongs to a large family of paralogs. To illustrate the problem, we have undertaken a genomewide analysis of interactions of members of the PE and PPE protein families of Mycobacterium tuberculosis. Our computational method uses structural information, operon organization, and protein coevolution to infer the interaction of PE and PPE proteins. Some 289 PE/PPE complexes were predicted out of a possible 5,590 PE/PPE pairs genomewide. Thirty-five of these predicted complexes were also found to have correlated mRNA expression, providing additional evidence for these interactions. We show that our method is applicable to other protein families, by analyzing interactions of the Esx family of proteins. Our resulting set of predictions is a starting point for genomewide experimental interaction screens of the PE and PPE families, and our method may be generally useful for detecting interactions of proteins within families having many paralogs.

  • Miller CS, Eisenberg D. (2008). Using inferred residue contacts to distinguish between correct and incorrect protein models.Bioinformatics. Jul 2008. 24(14):1575-82. [Abstract]

    The de novo prediction of 3D protein structure is enjoying a period of dramatic improvements. Often, a remaining difficulty is to select the model closest to the true structure from a group of low-energy candidates. To what extent can inter-residue contact predictions from multiple sequence alignments, information which is orthogonal to that used in most structure prediction algorithms, be used to identify those models most similar to the native protein structure?

  • Cruz D, Watson AD, Miller CS, Montoya D, Ochoa MT, Sieling PA, Gutierrez MA, Navab M, Reddy ST, Witztum JL, Fogelman AM, Rea TH, Eisenberg D, Berliner J, Modlin RL. (2008). Host-derived oxidized phospholipids and HDL regulate innate immunity in human leprosy.J. Clin. Invest.. Aug 2008. 118(8):2917-28. [Abstract]

    Intracellular pathogens survive by evading the host immune system and accessing host metabolic pathways to obtain nutrients for their growth. Mycobacterium leprae, the causative agent of leprosy, is thought to be the mycobacterium most dependent on host metabolic pathways, including host-derived lipids. Although fatty acids and phospholipids accumulate in the lesions of individuals with the lepromatous (also known as disseminated) form of human leprosy (L-lep), the origin and significance of these lipids remains unclear. Here we show that in human L-lep lesions, there was preferential expression of host lipid metabolism genes, including a group of phospholipases, and that these genes were virtually absent from the mycobacterial genome. Host-derived oxidized phospholipids were detected in macrophages within L-lep lesions, and 1 specific oxidized phospholipid, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC), accumulated in macrophages infected with live mycobacteria. Mycobacterial infection and host-derived oxidized phospholipids both inhibited innate immune responses, and this inhibition was reversed by the addition of normal HDL, a scavenger of oxidized phospholipids, but not by HDL from patients with L-lep. The accumulation of host-derived oxidized phospholipids in L-lep lesions is strikingly similar to observations in atherosclerosis, which suggests that the link between host lipid metabolism and innate immunity contributes to the pathogenesis of both microbial infection and metabolic disease.

  • Wang L, Maji SK, Sawaya MR, Eisenberg D, Riek R. (2008). Bacterial inclusion bodies contain amyloid-like structure.PLoS Biol.. Aug 2008. 6(8):e195. [Abstract]

    Protein aggregation is a process in which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as amorphous, lacking any long-range order, or highly ordered fibrils. Protein fibrils can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or can be reorganized beta-sheet-rich aggregates, termed amyloid-like fibrils. Amyloid fibrils are associated with several pathological conditions in humans, including Alzheimer disease and diabetes type II. We studied the structure of bacterial inclusion bodies, which have been believed to belong to the amorphous class of aggregates. We demonstrate that all three in vivo-derived inclusion bodies studied are amyloid-like and comprised of amino-acid sequence-specific cross-beta structure. These findings suggest that inclusion bodies are structured, that amyloid formation is an omnipresent process both in eukaryotes and prokaryotes, and that amino acid sequences evolve to avoid the amyloid conformation.

  • Sawaya MR, Wojtowicz WM, Andre I, Qian B, Wu W, Baker D, Eisenberg D, Zipursky SL. (2008). A double S shape provides the structural basis for the extraordinary binding specificity of Dscam isoforms.Cell. Sep 2008. 134(6):1007-18. [Abstract]

    Drosophila Dscam encodes a vast family of immunoglobulin (Ig)-containing proteins that exhibit isoform-specific homophilic binding. This diversity is essential for cell recognition events required for wiring the brain. Each isoform binds to itself but rarely to other isoforms. Specificity is determined by “matching” of three variable Ig domains within an approximately 220 kD ectodomain. Here, we present the structure of the homophilic binding region of Dscam, comprising the eight N-terminal Ig domains (Dscam(1-8)). Dscam(1-8) forms a symmetric homodimer of S-shaped molecules. This conformation, comprising two reverse turns, allows each pair of the three variable domains to “match” in an antiparallel fashion. Structural, genetic, and biochemical studies demonstrate that, in addition to variable domain “matching,” intramolecular interactions between constant domains promote homophilic binding. These studies provide insight into how “matching” at all three pairs of variable domains in Dscam mediates isoform-specific recognition.

  • Wiltzius JJ, Sievers SA, Sawaya MR, Cascio D, Popov D, Riekel C, Eisenberg D. (2008). Atomic structure of the cross-beta spine of islet amyloid polypeptide (amylin).Protein Sci.. Sep 2008. 17(9):1467-74. [Abstract]

    Human islet amyloid polypeptide (IAPP or amylin) is a 37-residue hormone found as fibrillar deposits in pancreatic extracts of nearly all type II diabetics. Although the cellular toxicity of IAPP has been established, the structure of the fibrillar form found in these deposits is unknown. Here we have crystallized two segments from IAPP, which themselves form amyloid-like fibrils. The atomic structures of these two segments, NNFGAIL and SSTNVG, were determined, and form the basis of a model for the most commonly observed, full-length IAPP polymorph.

  • Guo Z, Eisenberg D. (2008). The structure of a fibril-forming sequence, NNQQNY, in the context of a globular fold.Protein Sci.. Sep 2008. 17(9):1617-23. [Abstract]

    Numerous human disorders are associated with the formation of protein fibrils. The fibril-forming capacity of a protein has been found in recent studies to be determined by a short segment of residues that forms a dual beta-sheet, called a steric zipper, in the spine of the fibril. The question arises as to whether a fibril-forming segment, when inserted within the sequence of a globular protein, will invariably cause the protein to form fibrils. Here we investigate this question by inserting the known fibril-forming segment NNQQNY into the globular enzyme, T7 endonuclease I. From earlier studies, we know that in its fibril form, NNQQNY is in an extended conformation. We first found that the inserted NNQQNY stimulates fibril formation of T7 endonuclease I in solution. Thus NNQQNY within T7 endonuclease I can exist in an extended conformation, capable of forming the steric zipper in the core of a fibril. We also found that T7 endonuclease I folds into a decamer that does not form fibrils. We determined the structure of the decamer by X-ray crystallography, finding an unusual oligomer without point group symmetry, and finding that the NNQQNY segments within the decamer adopt two twisted conformations, neither is apparently able to fibrillize. We conclude that twisting of fibril forming sequences from the fully extended conformation, imposed by the context of their placement in proteins, can interfere with fibril formation.

  • Llewellyn R, Eisenberg DS. (2008). Annotating proteins with generalized functional linkages.Proc. Natl. Acad. Sci. U.S.A.. Nov 2008. 105(46):17700-5. [Abstract]

    As genome sequencing outstrips the rate of high-quality, low-throughput biochemical and genetic experimentation, accurate annotation of protein function becomes a bottleneck in the progress of the biomolecular sciences. Most gene products are now annotated by homology, in which an experimentally determined function is applied to a similar sequence. This procedure becomes error-prone between more divergent sequences and can contaminate biomolecular databases. Here, we propose a computational method of assignment of function, termed Generalized Functional Linkages (GFL), that combines nonhomology-based methods with other types of data. Functional linkages describe pairwise relationships between proteins that work together to perform a biological task. GFL provides a Bayesian framework that improves annotation by arbitrating a competition among biological process annotations to best describe the target protein. GFL addresses the unequal strengths of functional linkages among proteins, the quality of existing annotations, and the similarity among them while incorporating available knowledge about the cellular location or individual molecular function of the target protein. We demonstrate GFL with functional linkages defined by an algorithm known as zorch that quantifies connectivity in protein-protein interaction networks. Even when using proteins linked only by indirect or high-throughput interactions, GFL predicts the biological processes of many proteins in Saccharomyces cerevisiae, improving the accuracy of annotation by 20% over majority voting.

  • Zhang K, Sawaya MR, Eisenberg DS, Liao JC. (2008). Expanding metabolism for biosynthesis of nonnatural alcohols.Proc. Natl. Acad. Sci. U.S.A.. Dec 2008. 105(52):20653-8. [Abstract]

    Nature uses a limited set of metabolites to perform all of the biochemical reactions. To increase the metabolic capabilities of biological systems, we have expanded the natural metabolic network, using a nonnatural metabolic engineering approach. The branched-chain amino acid pathways are extended to produce abiotic longer chain keto acids and alcohols by engineering the chain elongation activity of 2-isopropylmalate synthase and altering the substrate specificity of downstream enzymes through rational protein design. When introduced into Escherichia coli, this nonnatural biosynthetic pathway produces various long-chain alcohols with carbon number ranging from 5 to 8. In particular, we demonstrate the feasibility of this approach by optimizing the biosynthesis of the 6-carbon alcohol, (S)-3-methyl-1-pentanol. This work demonstrates an approach to build artificial metabolism beyond the natural metabolic network. Nonnatural metabolites such as long chain alcohols are now included in the metabolite family of living systems.

  • Medrano-Soto A, Pal D, Eisenberg D. (2008). Inferring molecular function: contributions from functional linkages.Trends Genet.. Dec 2008. 24(12):587-90. [Abstract]

    In the current era of high-throughput sequencing and structure determination, functional annotation has become a bottleneck in biomedical science. Here, we show that automated inference of molecular function using functional linkages among genes increases the accuracy of functional assignments by > or =8% and enriches functional descriptions in > or =34% of top assignments. Furthermore, biochemical literature supports >80% of automated inferences for previously unannotated proteins. These results emphasize the benefit of incorporating functional linkages in protein annotation.


  • Strong M, Eisenberg D. (2007). The protein network as a tool for finding novel drug targets.Prog Drug Res. 2007. 64:191, 193-215. [Abstract]

    Proteins are often referred to as the molecular workhorses of the cell since they are responsible for the majority of functions within a living cell. From the generation of energy, to the replication of DNA, proteins play a central role in most cellular functions. Because of their importance to cellular viability, proteins are commonly the target of therapeutic drugs, ranging from antimicrobial to anticancer drugs. With the rise of drug resistant and multi-drug resistant forms of many diseases, it has become increasingly important to develop new strategies to identify alternative drug targets. One such strategy arises from the analysis of protein networks. Protein networks help define individual proteins within the context of all other cellular proteins. In this chapter we discuss methods for the identification and analysis of genome-wide protein networks, and discuss how protein networks can be used to aid the identification of novel drug targets.

  • Goulding CW, Bowers PM, Segelke B, Lekin T, Kim CY, Terwilliger TC, Eisenberg D. (2007). The structure and computational analysis of Mycobacterium tuberculosis protein CitE suggest a novel enzymatic function.J. Mol. Biol.. Jan 2007. 365(2):275-83. [Abstract]

    Fatty acid biosynthesis is essential for the survival of Mycobacterium tuberculosis and acetyl-coenzyme A (acetyl-CoA) is an essential precursor in this pathway. We have determined the 3-D crystal structure of M. tuberculosis citrate lyase beta-subunit (CitE), which as annotated should cleave protein bound citryl-CoA to oxaloacetate and a protein-bound CoA derivative. The CitE structure has the (beta/alpha)(8) TIM barrel fold with an additional alpha-helix, and is trimeric. We have determined the ternary complex bound with oxaloacetate and magnesium, revealing some of the conserved residues involved in catalysis. While the bacterial citrate lyase is a complex with three subunits, the M. tuberculosis genome does not contain the alpha and gamma subunits of this complex, implying that M. tuberculosis CitE acts differently from other bacterial CitE proteins. The analysis of gene clusters containing the CitE protein from 168 fully sequenced organisms has led us to identify a grouping of functionally related genes preserved in M. tuberculosis, Rattus norvegicus, Homo sapiens, and Mus musculus. We propose a novel enzymatic function for M. tuberculosis CitE in fatty acid biosynthesis that is analogous to bacterial citrate lyase but producing acetyl-CoA rather than a protein-bound CoA derivative.

  • Tsemekhman K, Goldschmidt L, Eisenberg D, Baker D. (2007). Cooperative hydrogen bonding in amyloid formation.Protein Sci.. Apr 2007. 16(4):761-4. [Abstract]

    Amyloid diseases, including Alzheimer’s and prion diseases, are each associated with unbranched protein fibrils. Each fibril is made of a particular protein, yet they share common properties. One such property is nucleation-dependent fibril growth. Monomers of amyloid-forming proteins can remain in dissolved form for long periods, before rapidly assembly into fibrils. The lag before growth has been attributed to slow kinetics of formation of a nucleus, on which other molecules can deposit to form the fibril. We have explored the energetics of fibril formation, based on the known molecular structure of a fibril-forming peptide from the yeast prion, Sup35, using both classical and quantum (density functional theory) methods. We find that the energetics of fibril formation for the first three layers are cooperative using both methods. This cooperativity is consistent with the observation that formation of amyloid fibrils involves slow nucleation and faster growth.

  • Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, Apostol MI, Thompson MJ, Balbirnie M, Wiltzius JJ, McFarlane HT, Madsen AØ, Riekel C, Eisenberg D. (2007). Atomic structures of amyloid cross-beta spines reveal varied steric zippers.Nature. May 2007. 447(7143):453-7. [Abstract]

    Amyloid fibrils formed from different proteins, each associated with a particular disease, contain a common cross-beta spine. The atomic architecture of a spine, from the fibril-forming segment GNNQQNY of the yeast prion protein Sup35, was recently revealed by X-ray microcrystallography. It is a pair of beta-sheets, with the facing side chains of the two sheets interdigitated in a dry ‘steric zipper’. Here we report some 30 other segments from fibril-forming proteins that form amyloid-like fibrils, microcrystals, or usually both. These include segments from the Alzheimer’s amyloid-beta and tau proteins, the PrP prion protein, insulin, islet amyloid polypeptide (IAPP), lysozyme, myoglobin, alpha-synuclein and beta(2)-microglobulin, suggesting that common structural features are shared by amyloid diseases at the molecular level. Structures of 13 of these microcrystals all reveal steric zippers, but with variations that expand the range of atomic architectures for amyloid-like fibrils and offer an atomic-level hypothesis for the basis of prion strains.

  • Guo Z, Eisenberg D. (2007). The mechanism of the amyloidogenic conversion of T7 endonuclease I.J. Biol. Chem.. May 2007. 282(20):14968-74. [Abstract]

    Amyloid fibrils are associated with a range of human disorders. Understanding the conversion of amyloidogenic proteins from their soluble forms to amyloid fibrils is critical for developing effective therapeutics. Previously we showed that T7 endonuclease I forms amyloid-like fibrils. Here we study the mechanism of the amyloidogenic conversion of T7 endonuclease I. We show that T7 endonuclease I forms fibrils at pH 6.8, but not at pH 6.0 or 8.0. The amyloidogenicity at pH 6.8 is not correlated with thermodynamic stability, unfolding cooperativity, or solubility. Thermal melting experiments at various pH values show that the protein has a distinctive thermal transition at pH 6.8. The transition at pH 6.8 has a lower transition temperature than the unfolding transitions observed at pH 6.0 and 8.0 and leads to a beta-rich conformation instead of an unfolded state. Electron microscopy shows that the thermal transition at pH 6.8 results in fibril formation. The thermal transition at pH 6.8 leads to a protein state that is not accessible at pH 6.0 or 8.0, showing that the existence of the amyloidogenic conformation of T7 endonuclease I depends sensitively on solution conditions. Therefore, we propose that fibrillizing proteins need to be “prepared” for fibrillization. Preparation may consist of amino acid replacements or changing solution conditions and may require retention of some aspects of native structure. In this model, some amyloid-enhancing mutations decrease protein stability, whereas others have little effect.

  • Goldschmidt L, Cooper DR, Derewenda ZS, Eisenberg D. (2007). Toward rational protein crystallization: A Web server for the design of crystallizable protein variants.Protein Sci.. Aug 2007. 16(8):1569-76. [Abstract]

    Growing well-diffracting crystals constitutes a serious bottleneck in structural biology. A recently proposed crystallization methodology for “stubborn crystallizers” is to engineer surface sequence variants designed to form intermolecular contacts that could support a crystal lattice. This approach relies on the concept of surface entropy reduction (SER), i.e., the replacement of clusters of flexible, solvent-exposed residues with residues with lower conformational entropy. This strategy minimizes the loss of conformational entropy upon crystallization and renders crystallization thermodynamically favorable. The method has been successfully used to crystallize more than 15 novel proteins, all stubborn crystallizers. But the choice of suitable sites for mutagenesis is not trivial. Herein, we announce a Web server, the surface entropy reduction prediction server (SERp server), designed to identify mutations that may facilitate crystallization. Suggested mutations are predicted based on an algorithm incorporating a conformational entropy profile, a secondary structure prediction, and sequence conservation. Minor considerations include the nature of flanking residues and gaps between mutation candidates. While designed to be used with default values, the server has many user-controlled parameters allowing for considerable flexibility. Within, we discuss (1) the methodology of the server, (2) how to interpret the results, and (3) factors that must be considered when selecting mutations. We also attempt to benchmark the server by comparing the server’s predictions with successful SER structures. In most cases, the structure yielding mutations were easily identified by the SERp server. The server can be accessed at http://www.doe-mbi.ucla.edu/Services/SER.

  • Salwinski L, Eisenberg D. (2007). The MiSink Plugin: Cytoscape as a graphical interface to the Database of Interacting Proteins.Bioinformatics. Aug 2007. 23(16):2193-5. [Abstract]

    The MiSink Plugin converts Cytoscape, an open-source bioinformatics platform for network visualization, to a graphical interface for the database of interacting proteins (DIP: http://dip.doe-mbi.ucla.edu). Seamless integration is possible by providing bi-directional communication between Cytoscape and any Web site supplying data in XML or tab-delimited format. Availability: MiSink is freely available for download at http://dip.doe-mbi.ucla.edu/Software.cgi.

  • Kim SM, Bowers PM, Pal D, Strong M, Terwilliger TC, Kaufmann M, Eisenberg D. (2007). Functional linkages can reveal protein complexes for structure determination.Structure. Sep 2007. 15(9):1079-89. [Abstract]

    In the study of protein complexes, is there a computational method for inferring which combinations of proteins in an organism are likely to form a crystallizable complex? Here we attempt to answer this question, using the Protein Data Bank (PDB) to assess the usefulness of inferred functional protein linkages from the Prolinks database. We find that of the 242 nonredundant prokaryotic protein complexes shared between the current PDB and Prolinks, 44% (107/242) contain proteins linked at high confidence by one or more methods of computed functional linkages. Similarly, high-confidence linkages detect 47% of known Escherichia coli protein complexes, with 45% accuracy. Together these findings suggest that functional linkages will be useful in defining protein complexes for structural studies, including for structural genomics. We offer a database of inferred linkages corresponding to likely protein complexes for some 629,952 pairs of proteins in 154 prokaryotes and archaea.

  • Anderson DH, Kickhoefer VA, Sievers SA, Rome LH, Eisenberg D. (2007). Draft crystal structure of the vault shell at 9-A resolution.PLoS Biol.. Nov 2007. 5(11):e318. [Abstract]

    Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-A resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules.

  • Kuriyan J, Eisenberg D. (2007). The origin of protein interactions and allostery in colocalization.Nature. Dec 2007. 450(7172):983-90. [Abstract]

    Two fundamental principles can account for how regulated networks of interacting proteins originated in cells. These are the law of mass action, which holds that the binding of one molecule to another increases with concentration, and the fact that the colocalization of molecules vastly increases their local concentrations. It follows that colocalization can amplify the effect on one protein of random mutations in another protein and can therefore, through natural selection, lead to interactions between proteins and to a startling variety of complex allosteric controls. It also follows that allostery is common and that homologous proteins can have different allosteric mechanisms. Thus, the regulated protein networks of organisms seem to be the inevitable consequence of natural selection operating under physical laws.


  • Nelson R, Eisenberg D. (2006). Structural models of amyloid-like fibrils.Adv. Protein Chem.. 2006. 73:235-82. [Abstract]

    Amyloid fibrils are elongated, insoluble protein aggregates deposited in vivo in amyloid diseases, and amyloid-like fibrils are formed in vitro from soluble proteins. Both of these groups of fibrils, despite differences in the sequence and native structure of their component proteins, share common properties, including their core structure. Multiple models have been proposed for the common core structure, but in most cases, atomic-level structural details have yet to be determined. Here we review several structural models proposed for amyloid and amyloid-like fibrils and relate features of these models to the common fibril properties. We divide models into three classes: Refolding, Gain-of-Interaction, and Natively Disordered. The Refolding models propose structurally distinct native and fibrillar states and suggest that backbone interactions drive fibril formation. In contrast, the Gain-of-Interaction models propose a largely native-like structure for the protein in the fibril and highlight the importance of specific sequences in fibril formation. The Natively Disordered models have aspects in common with both Refolding and Gain-of-Interaction models. While each class of model suggests explanations for some of the common fibril properties, and some models, such as Gain-of-Interaction models with a cross-beta spine, fit a wider range of properties than others, no one class provides a complete explanation for all amyloid fibril behavior.

  • Wang S, Eisenberg D. (2006). Crystal structure of the pantothenate synthetase from Mycobacterium tuberculosis, snapshots of the enzyme in action.Biochemistry. Feb 2006. 45(6):1554-61. [Abstract]

    Pantothenate synthetase (PS) from Mycobacterium tuberculosis represents a potential target for antituberculosis drugs. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Previously, we determined the crystal structure of PS from M. tuberculosis and its complexes with AMPCPP, pantoate, and pantoyl adenylate. Here, we describe the crystal structure of this enzyme complexed with AMP and its last substrate, beta-alanine, and show that the phosphate group of AMP serves as an anchor for the binding of beta-alanine. This structure confirms that binding of beta-alanine in the active site cavity can occur only after formation of the pantoyl adenylate intermediate. A new crystal form was also obtained; it displays the flexible wall of the active site cavity in a conformation incapable of binding pantoate. Soaking of this crystal form with ATP and pantoate gives a fully occupied complex of PS with ATP. Crystal structures of these complexes with substrates, the reaction intermediate, and the reaction product AMP provide a step-by-step view of the PS-catalyzed reaction. A detailed reaction mechanism and its implications for inhibitor design are discussed.

  • Ivanova MI, Thompson MJ, Eisenberg D. (2006). A systematic screen of beta(2)-microglobulin and insulin for amyloid-like segments.Proc. Natl. Acad. Sci. U.S.A.. Mar 2006. 103(11):4079-82. [Abstract]

    Identifying sequence determinants of fibril-forming proteins is crucial for understanding the processes causing >20 proteins to form pathological amyloid depositions. Our approach to identifying which sequences form amyloid-like fibrils is to screen the amyloid-forming proteins human insulin and beta(2)-microglobulin for segments that form fibrils. Our screen is of 60 sequentially overlapping peptides, 59 being six residues in length and 1 being five residues, covering every noncysteine-containing segment in these two proteins. Each peptide was characterized as amyloid-like or nonfibril-forming. Amyloid-like peptides formed fibrils visible in electron micrographs or needle-like microcrystals showing a cross-beta diffraction pattern. Eight of the 60 peptides (three from insulin and five from beta(2)-microglobulin) were identified as amyloid-like. The results of the screen were used to assess the computational method, and good agreement between prediction and experiments was found. This agreement suggests that the pair-of-sheets, zipper spine model on which the computational method is based is at least approximately correct for the structure of the fibrils and suggests the nature of the sequence signal for formation of amyloid-like fibrils.

  • Thompson MJ, Sievers SA, Karanicolas J, Ivanova MI, Baker D, Eisenberg D. (2006). The 3D profile method for identifying fibril-forming segments of proteins.Proc. Natl. Acad. Sci. U.S.A.. Mar 2006. 103(11):4074-8. [Abstract]

    Based on the crystal structure of the cross-beta spine formed by the peptide NNQQNY, we have developed a computational approach for identifying those segments of amyloidogenic proteins that themselves can form amyloid-like fibrils. The approach builds on experiments showing that hexapeptides are sufficient for forming amyloid-like fibrils. Each six-residue peptide of a protein of interest is mapped onto an ensemble of templates, or 3D profile, generated from the crystal structure of the peptide NNQQNY by small displacements of one of the two intermeshed beta-sheets relative to the other. The energy of each mapping of a sequence to the profile is evaluated by using ROSETTADESIGN, and the lowest energy match for a given peptide to the template library is taken as the putative prediction. If the energy of the putative prediction is lower than a threshold value, a prediction of fibril formation is made. This method can reach an accuracy of approximately 80% with a P value of approximately 10(-12) when a conservative energy threshold is used to separate peptides that form fibrils from those that do not. We see enrichment for positive predictions in a set of fibril-forming segments of amyloid proteins, and we illustrate the method with applications to proteins of interest in amyloid research.

  • Eisenberg D, Marcotte E, McLachlan AD, Pellegrini M. (2006). Bioinformatic challenges for the next decade(s).Philos. Trans. R. Soc. Lond., B, Biol. Sci.. Mar 2006. 361(1467):525-7. [Abstract]

    The science of bioinformatics has developed in the wake of methods to determine the sequences of the informational macromolecules–DNAs, RNAs and proteins. But in a wider sense, the biological world depends in its every process on the transmission of information, and hence bioinformatics is the fundamental core of biology. We here give a consideration of some of the key problems of bioinformatics in the coming decade, and perhaps longer.

  • Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, Ochoa MT, Schauber J, Wu K, Meinken C, Kamen DL, Wagner M, Bals R, Steinmeyer A, Zügel U, Gallo RL, Eisenberg D, Hewison M, Hollis BW, Adams JS, Bloom BR, Modlin RL. (2006). Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response.Science. Mar 2006. 311(5768):1770-3. [Abstract]

    In innate immune responses, activation of Toll-like receptors (TLRs) triggers direct antimicrobial activity against intracellular bacteria, which in murine, but not human, monocytes and macrophages is mediated principally by nitric oxide. We report here that TLR activation of human macrophages up-regulated expression of the vitamin D receptor and the vitamin D-1-hydroxylase genes, leading to induction of the antimicrobial peptide cathelicidin and killing of intracellular Mycobacterium tuberculosis. We also observed that sera from African-American individuals, known to have increased susceptibility to tuberculosis, had low 25-hydroxyvitamin D and were inefficient in supporting cathelicidin messenger RNA induction. These data support a link between TLRs and vitamin D-mediated innate immunity and suggest that differences in ability of human populations to produce vitamin D may contribute to susceptibility to microbial infection.

  • Nelson R, Eisenberg D. (2006). Recent atomic models of amyloid fibril structure.Curr. Opin. Struct. Biol.. Apr 2006. 16(2):260-5. [Abstract]

    Despite the difficulties associated with determining atomic-level structures for materials that are fibrous, structural biologists are making headway in understanding the architecture of amyloid-like fibrils. It has long been recognized that these fibrils contain a cross-beta spine, with beta-strands perpendicular to the fibril axis. Recently, atomic structures have been determined for some of these cross-beta spines, revealing a pair of beta-sheets mated closely together by intermeshing sidechains in what has been termed a steric zipper. To explain the conversion of proteins from soluble to fibrous forms, several types of models have been proposed: refolding, natively disordered and gain of interaction. The gain-of-interaction models may additionally be subdivided into direct stacking, cross-beta spine, three-dimensional domain swapping and three-dimensional domain swapping with a cross-beta spine.

  • Guo Z, Eisenberg D. (2006). Runaway domain swapping in amyloid-like fibrils of T7 endonuclease I.Proc. Natl. Acad. Sci. U.S.A.. May 2006. 103(21):8042-7. [Abstract]

    Amyloid fibrils are associated with >20 fatal human disorders, including Alzheimer’s, Parkinson’s, and prion diseases. Knowledge of how soluble proteins assemble into amyloid fibrils remains elusive despite its potential usefulness for developing diagnostics and therapeutics. In at least some fibrils, runaway domain swapping has been proposed as a possible mechanism for fibril formation. In runaway domain swapping, each protein molecule swaps a domain into the complementary domain of the adjacent molecule along the fibril. Here we show that T7 endonuclease I, a naturally domain-swapped dimeric protein, can form amyloid-like fibrils. Using protein engineering, we designed a double-cysteine mutant that forms amyloid-like fibrils in which molecules of T7 endonuclease I are linked by intermolecular disulfide bonds. Because the disulfide bonds are designed to form only at the domain-swapped dimer interface, the resulting covalently linked fibrils show that T7 endonuclease I forms fibrils by a runaway domain swap. In addition, we show that the disulfide mutant exists in two conformations, only one of which is able to form fibrils. We also find that domain-swapped dimers, if locked in a close-ended dimeric form, are unable to form fibrils. Our study provides strong evidence for runaway domain swapping in the formation of an amyloid-like fibril and, consequently, a molecular explanation for specificity and stability of fibrils. In addition, our results suggest that inhibition of fibril formation for domain-swapped proteins may be achieved by stabilizing domain-swapped dimers.

  • Bennett MJ, Sawaya MR, Eisenberg D. (2006). Deposition diseases and 3D domain swapping.Structure. May 2006. 14(5):811-24. [Abstract]

    Protein aggregation is a feature of both normal cellular assemblies and pathological protein depositions. Although the limited order of aggregates has often impeded their structural characterization, 3D domain swapping has been implicated in the formation of several protein aggregates. Here, we review known structures displaying 3D domain swapping in the context of amyloid and related fibrils, prion proteins, and macroscopic aggregates, and we discuss the possible involvement of domain swapping in protein deposition diseases.

  • Strong M, Sawaya MR, Wang S, Phillips M, Cascio D, Eisenberg D. (2006). Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis.Proc. Natl. Acad. Sci. U.S.A.. May 2006. 103(21):8060-5. [Abstract]

    The developing science called structural genomics has focused to date mainly on high-throughput expression of individual proteins, followed by their purification and structure determination. In contrast, the term structural biology is used to denote the determination of structures, often complexes of several macromolecules, that illuminate aspects of biological function. Here we bridge structural genomics to structural biology with a procedure for determining protein complexes of previously unknown function from any organism with a sequenced genome. From computational genomic analysis, we identify functionally linked proteins and verify their interaction in vitro by coexpression/copurification. We illustrate this procedure by the structural determination of a previously unknown complex between a PE and PPE protein from the Mycobacterium tuberculosis genome, members of protein families that constitute approximately 10% of the coding capacity of this genome. The predicted complex was readily expressed, purified, and crystallized, although we had previously failed in expressing individual PE and PPE proteins on their own. The reason for the failure is clear from the structure, which shows that the PE and PPE proteins mate along an extended apolar interface to form a four-alpha-helical bundle, where two of the alpha-helices are contributed by the PE protein and two by the PPE protein. Our entire procedure for the identification, characterization, and structural determination of protein complexes can be scaled to a genome-wide level.

  • Eisenberg D, Nelson R, Sawaya MR, Balbirnie M, Sambashivan S, Ivanova MI, Madsen AØ, Riekel C. (2006). The structural biology of protein aggregation diseases: Fundamental questions and some answers.Acc. Chem. Res.. Sep 2006. 39(9):568-75. [Abstract]

    Amyloid fibrils are found in association with at least two dozen fatal diseases. The tendency of numerous proteins to convert into amyloid-like fibrils poses fundamental questions for structural biology and for protein science in general. Among these are the following: What is the structure of the cross-beta spine, common to amyloid-like fibrils? Is there a sequence signature for proteins that form amyloid-like fibrils? What is the nature of the structural conversion from native to amyloid states, and do fibril-forming proteins have two distinct stable states, the native state and the amyloid state? What is the basis of protein complementarity, in which a protein chain can bind to itself? We offer tentative answers here, based on our own recent structural studies.


  • Riley R, Lee C, Sabatti C, Eisenberg D. (2005). Inferring protein domain interactions from databases of interacting proteins.Genome Biol.. 2005. 6(10):R89. [Abstract]

    We describe domain pair exclusion analysis (DPEA), a method for inferring domain interactions from databases of interacting proteins. DPEA features a log odds score, Eij, reflecting confidence that domains i and j interact. We analyzed 177,233 potential domain interactions underlying 26,032 protein interactions. In total, 3,005 high-confidence domain interactions were inferred, and were evaluated using known domain interactions in the Protein Data Bank. DPEA may prove useful in guiding experiment-based discovery of previously unrecognized domain interactions.

  • Pal D, Eisenberg D. (2005). Inference of protein function from protein structure.Structure. Jan 2005. 13(1):121-30. [Abstract]

    Structural genomics has brought us three-dimensional structures of proteins with unknown functions. To shed light on such structures, we have developed ProKnow (http://www.doe-mbi.ucla.edu/Services/ProKnow/), which annotates proteins with Gene Ontology functional terms. The method extracts features from the protein such as 3D fold, sequence, motif, and functional linkages and relates them to function via the ProKnow knowledgebase of features, which links features to annotated functions via annotation profiles. Bayes’ theorem is used to compute weights of the functions assigned, using likelihoods based on the extracted features. The description level of the assigned function is quantified by the ontology depth (from 1 = general to 9 = specific). Jackknife tests show approximately 89% correct assignments at ontology depth 1 and 40% at depth 9, with 93% coverage of 1507 distinct folded proteins. Overall, about 70% of the assignments were inferred correctly. This level of performance suggests that ProKnow is a useful resource in functional assessments of novel proteins.

  • Li H, Pellegrini M, Eisenberg D. (2005). Detection of parallel functional modules by comparative analysis of genome sequences.Nat. Biotechnol.. Feb 2005. 23(2):253-60. [Abstract]

    Parallel functional modules are separate sets of proteins in an organism that catalyze the same or similar biochemical reactions but act on different substrates or use different cofactors. They originate by gene duplication during evolution. Parallel functional modules provide versatility and complexity to organisms, and increase cellular flexibility and robustness. We have developed a four-step approach for genome-wide discovery of parallel modules from protein functional linkages. From ten genomes, we identified 37 cellular systems that consist of parallel functional modules. This approach recovers known parallel complexes and pathways, and discovers new ones that conventional homology-based methods did not previously reveal, as illustrated by examples of peptide transporters in Escherichia coli and nitrogenases in Rhodopseudomonas palustris. The approach untangles intertwined functional linkages between parallel functional modules and expands our ability to decode protein functions from genome sequences.

  • Müller P, Sawaya MR, Pashkov I, Chan S, Nguyen C, Wu Y, Perry LJ, Eisenberg D. (2005). The 1.70 angstroms X-ray crystal structure of Mycobacterium tuberculosis phosphoglycerate mutase.Acta Crystallogr. D Biol. Crystallogr.. Mar 2005. 61(Pt 3):309-15. [Abstract]

    The single-crystal X-ray structure of phosphoglycerate mutase from Mycobacterium tuberculosis has been determined at a resolution of 1.70 angstroms. The C-terminal tail of each of the subunits is flexible and disordered; however, for one of the four chains (chain A) all but five residues of the chain could be modeled. Noteworthy features of the structure include the active site and a proline-rich segment in each monomer forming a short left-handed polyprolyl helix. These segments lie on the enzyme surface and could conceivably participate in protein-protein interactions.

  • Li H, Sawaya MR, Tabita FR, Eisenberg D. (2005). Crystal structure of a RuBisCO-like protein from the green sulfur bacterium Chlorobium tepidum.Structure. May 2005. 13(5):779-89. [Abstract]

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the incorporation of atmospheric CO(2) into ribulose 1,5-bisphosphate (RuBP). RuBisCOs are classified into four forms based on sequence similarity: forms I, II and III are bona fide RuBisCOs; form IV, also called the RuBisCO-like protein (RLP), lacks several of the substrate binding and catalytic residues and does not catalyze RuBP-dependent CO(2) fixation in vitro. To contribute to understanding the function of RLPs, we determined the crystal structure of the RLP from Chlorobium tepidum. The overall structure of the RLP is similar to the structures of the three other forms of RuBisCO; however, the active site is distinct from those of bona fide RuBisCOs and suggests that the RLP is possibly capable of catalyzing enolization but not carboxylation. Bioinformatic analysis of the protein functional linkages suggests that this RLP coevolved with enzymes of the bacteriochlorophyll biosynthesis pathway and may be involved in processes related to photosynthesis.

  • Goulding CW, Apostol MI, Sawaya MR, Phillips M, Parseghian A, Eisenberg D. (2005). Regulation by oligomerization in a mycobacterial folate biosynthetic enzyme.J. Mol. Biol.. May 2005. 349(1):61-72. [Abstract]

    Folate derivatives are essential cofactors in the biosynthesis of purines, pyrimidines and amino acids across all forms of life. Mammals uptake folate from their diets, whereas most bacteria must synthesize folate de novo. Therefore, the enzymes in the folate biosynthetic pathway are attractive drug targets against bacterial pathogens such as Mycobacterium tuberculosis, the cause of the world’s most deadly infectious disease, tuberculosis (TB). M.tuberculosis 7,8-dihydroneopterin aldolase (Mtb FolB, DHNA) is the second enzyme in the folate biosynthetic pathway, which catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and glycoaldehyde. The 1.6A X-ray crystal structure of Mtb FolB complexed with its product, 6-hydroxymethyl-7,8-dihydropterin, reveals an octameric assembly similar to that seen in crystal structures of other FolB homologs. However, the 2.5A crystal structure of unliganded Mtb FolB reveals a novel tetrameric oligomerization state, with only partially formed active sites. A substrate induced conformational change appears to be necessary to convert the inactive tetramer to the active octamer. Ultracentrifugation confirmed that in solution unliganded Mtb FolB is mainly tetrameric and upon addition of substrate FolB is predominantly octameric. Kinetic analysis of substrate binding gives a Hill coefficient of 2.0, indicating positive cooperativity. We hypothesize that Mtb FolB displays cooperativity in substrate binding to regulate the cellular concentration of 7,8-dihydroneopterin, so that it may function not only as a precursor to folate but also as an antioxidant for the survival of M.tuberculosis against host defenses.

  • Nelson R, Sawaya MR, Balbirnie M, Madsen AØ, Riekel C, Grothe R, Eisenberg D. (2005). Structure of the cross-beta spine of amyloid-like fibrils.Nature. Jun 2005. 435(7043):773-8. [Abstract]

    Numerous soluble proteins convert to insoluble amyloid-like fibrils that have common properties. Amyloid fibrils are associated with fatal diseases such as Alzheimer’s, and amyloid-like fibrils can be formed in vitro. For the yeast protein Sup35, conversion to amyloid-like fibrils is associated with a transmissible infection akin to that caused by mammalian prions. A seven-residue peptide segment from Sup35 forms amyloid-like fibrils and closely related microcrystals, from which we have determined the atomic structure of the cross-beta spine. It is a double beta-sheet, with each sheet formed from parallel segments stacked in register. Side chains protruding from the two sheets form a dry, tightly self-complementing steric zipper, bonding the sheets. Within each sheet, every segment is bound to its two neighbouring segments through stacks of both backbone and side-chain hydrogen bonds. The structure illuminates the stability of amyloid fibrils, their self-seeding characteristic and their tendency to form polymorphic structures.

  • Sambashivan S, Liu Y, Sawaya MR, Gingery M, Eisenberg D. (2005). Amyloid-like fibrils of ribonuclease A with three-dimensional domain-swapped and native-like structure.Nature. Sep 2005. 437(7056):266-9. [Abstract]

    Amyloid or amyloid-like fibrils are elongated, insoluble protein aggregates, formed in vivo in association with neurodegenerative diseases or in vitro from soluble native proteins, respectively. The underlying structure of the fibrillar or ‘cross-beta’ state has presented long-standing, fundamental puzzles of protein structure. These include whether fibril-forming proteins have two structurally distinct stable states, native and fibrillar, and whether all or only part of the native protein refolds as it converts to the fibrillar state. Here we show that a designed amyloid-like fibril of the well-characterized enzyme RNase A contains native-like molecules capable of enzymatic activity. In addition, these functional molecular units are formed from a core RNase A domain and a swapped complementary domain. These findings are consistent with the zipper-spine model in which a cross-beta spine is decorated with three-dimensional domain-swapped functional units, retaining native-like structure.

  • Bowers PM, O’Connor BD, Cokus SJ, Sprinzak E, Yeates TO, Eisenberg D. (2005). Utilizing logical relationships in genomic data to decipher cellular processes.FEBS J.. Oct 2005. 272(20):5110-8. [Abstract]

    The wealth of available genomic data has spawned a corresponding interest in computational methods that can impart biological meaning and context to these experiments. Traditional computational methods have drawn relationships between pairs of proteins or genes based on notions of equality or similarity between their patterns of occurrence or behavior. For example, two genes displaying similar variation in expression, over a number of experiments, may be predicted to be functionally related. We have introduced a natural extension of these approaches, instead identifying logical relationships involving triplets of proteins. Triplets provide for various discrete kinds of logic relationships, leading to detailed inferences about biological associations. For instance, a protein C might be encoded within an organism if, and only if, two other proteins A and B are also both encoded within the organism, thus suggesting that gene C is functionally related to genes A and B. The method has been applied fruitfully to both phylogenetic and microarray expression data, and has been used to associate logical combinations of protein activity with disease state phenotypes, revealing previously unknown ternary relationships among proteins, and illustrating the inherent complexities that arise in biological data.


  • Bowers PM, Pellegrini M, Thompson MJ, Fierro J, Yeates TO, Eisenberg D. (2004). Prolinks: a database of protein functional linkages derived from coevolution.Genome Biol.. 2004. 5(5):R35. [Abstract]

    The advent of whole-genome sequencing has led to methods that infer protein function and linkages. We have combined four such algorithms (phylogenetic profile, Rosetta Stone, gene neighbor and gene cluster) in a single database–Prolinks–that spans 83 organisms and includes 10 million high-confidence links. The Proteome Navigator tool allows users to browse predicted linkage networks interactively, providing accompanying annotation from public databases. The Prolinks database and the Proteome Navigator tool are available for use online at http://dip.doe-mbi.ucla.edu/pronav.

  • Kleiger G, Panina EM, Mallick P, Eisenberg D. (2004). PFIT and PFRIT: bioinformatic algorithms for detecting glycosidase function from structure and sequence.Protein Sci.. Jan 2004. 13(1):221-9. [Abstract]

    The identification of the enzymes involved in the metabolism of simple and complex carbohydrates presents one bioinformatic challenge in the post-genomic era. Here, we present the PFIT and PFRIT algorithms for identifying those proteins adopting the alpha/beta barrel fold that function as glycosidases. These algorithms are based on the observation that proteins adopting the alpha/beta barrel fold share positions in their tertiary structures having equivalent sets of atomic interactions. These are conserved tertiary interaction positions, which have been implicated in both structure and function. Glycosidases adopting the alpha/beta barrel fold share more conserved tertiary interactions than alpha/beta barrel proteins having other functions. The enrichment pattern of conserved tertiary interactions in the glycosidases is the information that PFIT and PFRIT use to predict whether any given alpha/beta barrel will function as a glycosidase or not. Using as a test set a database of 19 glycosidase and 45 nonglycosidase alpha/beta barrel proteins with low sequence similarity, PFIT and PFRIT can correctly predict glycosidase function for 84% of the proteins known to function as glycosidases. PFIT and PFRIT incorrectly predict glycosidase function for 25% of the nonglycosidases. The program PSI-BLAST can also correctly identify 84% of the 19 glycosidases, however, it incorrectly predicts glycosidase function for 50% of the nonglycosidases (twofold greater than PFIT and PFRIT). Overall, we demonstrate that the structure-based PFIT and PFRIT algorithms are both more selective and sensitive for predicting glycosidase function than the sequence-based PSI-BLAST algorithm.

  • Salwinski L, Miller CS, Smith AJ, Pettit FK, Bowie JU, Eisenberg D. (2004). The Database of Interacting Proteins: 2004 update.Nucleic Acids Res.. Jan 2004. 32(Database issue):D449-51. [Abstract]

    The Database of Interacting Proteins (http://dip.doe-mbi.ucla.edu) aims to integrate the diverse body of experimental evidence on protein-protein interactions into a single, easily accessible online database. Because the reliability of experimental evidence varies widely, methods of quality assessment have been developed and utilized to identify the most reliable subset of the interactions. This CORE set can be used as a reference when evaluating the reliability of high-throughput protein-protein interaction data sets, for development of prediction methods, as well as in the studies of the properties of protein interaction networks.

  • Goulding CW, Apostol MI, Gleiter S, Parseghian A, Bardwell J, Gennaro M, Eisenberg D. (2004). Gram-positive DsbE proteins function differently from Gram-negative DsbE homologs. A structure to function analysis of DsbE from Mycobacterium tuberculosis.J. Biol. Chem.. Jan 2004. 279(5):3516-24. [Abstract]

    Mycobacterium tuberculosis, a Gram-positive bacterium, encodes a secreted Dsb-like protein annotated as Mtb DsbE (Rv2878c, also known as MPT53). Because Dsb proteins in Escherichia coli and other bacteria seem to catalyze proper folding during protein secretion and because folding of secreted proteins is thought to be coupled to disulfide oxidoreduction, the function of Mtb DsbE may be to ensure that secreted proteins are in their correctly folded states. We have determined the crystal structure of Mtb DsbE to 1.1 A resolution, which reveals a thioredoxin-like domain with a typical CXXC active site. These cysteines are in their reduced state. Biochemical characterization of Mtb DsbE reveals that this disulfide oxidoreductase is an oxidant, unlike Gram-negative bacteria DsbE proteins, which have been shown to be weak reductants. In addition, the pK(a) value of the active site, solvent-exposed cysteine is approximately 2 pH units lower than that of Gram-negative DsbE homologs. Finally, the reduced form of Mtb DsbE is more stable than the oxidized form, and Mtb DsbE is able to oxidatively fold hirudin. Structural and biochemical analysis implies that Mtb DsbE functions differently from Gram-negative DsbE homologs, and we discuss its possible functional role in the bacterium.

  • Ivanova MI, Sawaya MR, Gingery M, Attinger A, Eisenberg D. (2004). An amyloid-forming segment of beta2-microglobulin suggests a molecular model for the fibril.Proc. Natl. Acad. Sci. U.S.A.. Jul 2004. 101(29):10584-9. [Abstract]

    In humans suffering from dialysis-related amyloidosis, the protein beta2-microglobulin (beta2M) is deposited as an amyloid; however, an amyloid of beta2M is unknown in mice. beta2M sequences from human and mouse are 70% identical, but there is a seven-residue peptide in which six residues differ. This peptide from human beta2M forms amyloid in vitro, whereas the mouse peptide does not. Substitution of the human peptide for its counterpart in the mouse sequence results in the formation of amyloid in vitro. These results show that a seven-residue segment of human beta2M is sufficient to convert beta2M to the amyloid state, and that specific residue interactions are crucial to the conversion. These observations are consistent with a proposed Zipper-spine model for beta2M amyloid, in which the spine of the fibril consists of an anhydrous beta-sheet.

  • Salwinski L, Eisenberg D. (2004). In silico simulation of biological network dynamics.Nat. Biotechnol.. Aug 2004. 22(8):1017-9. [Abstract]

    Realistic simulation of biological networks requires stochastic simulation approaches because of the small numbers of molecules per cell. The high computational cost of stochastic simulation on conventional microprocessor-based computers arises from the intrinsic disparity between the sequential steps executed by a microprocessor program and the highly parallel nature of information flow within biochemical networks. This disparity is reduced with the Field Programmable Gate Array (FPGA)-based approach presented here. The parallel architecture of FPGAs, which can simulate the basic reaction steps of biological networks, attains simulation rates at least an order of magnitude greater than currently available microprocessors.

  • Reinhardt A, Eisenberg D. (2004). DPANN: improved sequence to structure alignments following fold recognition.Proteins. Aug 2004. 56(3):528-38. [Abstract]

    In fold recognition (FR) a protein sequence of unknown structure is assigned to the closest known three-dimensional (3D) fold. Although FR programs can often identify among all possible folds the one a sequence adopts, they frequently fail to align the sequence to the equivalent residue positions in that fold. Such failures frustrate the next step in structure prediction, protein model building. Hence it is desirable to improve the quality of the alignments between the sequence and the identified structure. We have used artificial neural networks (ANN) to derive a substitution matrix to create alignments between a protein sequence and a protein structure through dynamic programming (DPANN: Dynamic Programming meets Artificial Neural Networks). The matrix is based on the amino acid type and the secondary structure state of each residue. In a database of protein pairs that have the same fold but lack sequences-similarity, DPANN aligns over 30% of all sequences to the paired structure, resembling closely the structural superposition of the pair. In over half of these cases the DPANN alignment is close to the structural superposition, although the initial alignment from the step of fold recognition is not close. Conversely, the alignment created during fold recognition outperforms DPANN in only 10% of all cases. Thus application of DPANN after fold recognition leads to substantial improvements in alignment accuracy, which in turn provides more useful templates for the modeling of protein structures. In the artificial case of using actual instead of predicted secondary structures for the probe protein, over 50% of the alignments are successful.

  • Bowers PM, Cokus SJ, Eisenberg D, Yeates TO. (2004). Use of logic relationships to decipher protein network organization.Science. Dec 2004. 306(5705):2246-9. [Abstract]

    A major focus of genome research is to decipher the networks of molecular interactions that underlie cellular function. We describe a computational approach for identifying detailed relationships between proteins on the basis of genomic data. Logic analysis of phylogenetic profiles identifies triplets of proteins whose presence or absence obey certain logic relationships. For example, protein C may be present in a genome only if proteins A and B are both present. The method reveals many previously unidentified higher order relationships. These relationships illustrate the complexities that arise in cellular networks because of branching and alternate pathways, and they also facilitate assignment of cellular functions to uncharacterized proteins.


  • Strong M, Mallick P, Pellegrini M, Thompson MJ, Eisenberg D. (2003). Inference of protein function and protein linkages in Mycobacterium tuberculosis based on prokaryotic genome organization: a combined computational approach.Genome Biol.. 2003. 4(9):R59. [Abstract]

    The genome of Mycobacterium tuberculosis was analyzed using recently developed computational approaches to infer protein function and protein linkages. We evaluated and employed a method to infer genes likely to belong to the same operon, as judged by the nucleotide distance between genes in the same genomic orientation, and combined this method with those of the Rosetta Stone, Phylogenetic Profile and conserved Gene Neighbor computational methods for the inference of protein function.

  • Eisenberg D. (2003). John T. Edsall as tutor and teacher.Biophys. Chem.. 2003. 100(1-3):91-3. [Abstract]
  • Anderson DH, Sawaya MR, Cascio D, Ernst W, Modlin R, Krensky A, Eisenberg D. (2003). Granulysin crystal structure and a structure-derived lytic mechanism.J. Mol. Biol.. Jan 2003. 325(2):355-65. [Abstract]

    Our crystal structure of granulysin suggests a mechanism for lysis of bacterial membranes by granulysin, a 74-residue basic protein from human cytolytic T lymphocyte and natural killer cells. We determined the initial crystal structure of selenomethionyl granulysin by MAD phasing at 2A resolution. We present the structure model refined using native diffraction data to 0.96A resolution. The five-helical bundle of granulysin resembles other “saposin folds” (such as NK-lysin). Positive charges distribute in a ring around the granulysin molecule, and one face has net positive charge. Sulfate ions bind near the segment of the molecule identified as most membrane-lytic and of highest hydrophobic moment. The ion locations may indicate granulysin’s orientation of initial approach towards the membrane. The crystal packing reveals one way to pack a sheet of granulysin molecules at the cell surface for a concerted lysis effort. The energy of binding granulysin charges to the bacterial membrane could drive the subsequent lytic processes. The loosely packed core facilitates a hinge or scissors motion towards exposure of hydrophobic surface that we propose tunnels the granulysin into the fracturing target membrane.

  • McCarty AS, Kleiger G, Eisenberg D, Smale ST. (2003). Selective dimerization of a C2H2 zinc finger subfamily.Mol. Cell. Feb 2003. 11(2):459-70. [Abstract]

    The C2H2 zinc finger is the most prevalent protein motif in the mammalian proteome. Two C2H2 fingers in Ikaros are dedicated to homotypic interactions between family members. We show here that these fingers comprise a bona fide dimerization domain. Dimerization is highly selective, however, as homologous domains from the TRPS-1 and Drosophila Hunchback proteins support homodimerization, but not heterodimerization with Ikaros. Ikaros-Hunchback selectivity is determined by 11 residues concentrated within the alpha-helical regions typically involved in base recognition. Preferential homodimerization of one chimeric protein predicts a parallel dimer interface and establishes the feasibility of creating novel dimer specificities. These results demonstrate that the C2H2 motif provides a versatile platform for both sequence-specific protein-nucleic acid interactions and highly specific dimerization.

  • Mura C, Katz JE, Clarke SG, Eisenberg D. (2003). Structure and function of an archaeal homolog of survival protein E (SurEalpha): an acid phosphatase with purine nucleotide specificity.J. Mol. Biol.. Mar 2003. 326(5):1559-75. [Abstract]

    The survival protein E (SurE) family was discovered by its correlation to stationary phase survival of Escherichia coli and various repair proteins involved in sustaining this and other stress-response phenotypes. In order to better understand this ancient and well-conserved protein family, we have determined the 2.0A resolution crystal structure of SurEalpha from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum (Pae). This first structure of an archaeal SurE reveals significant similarities to and differences from the only other known SurE structure, that from the eubacterium Thermatoga maritima (Tma). Both SurE monomers adopt similar folds; however, unlike the Tma SurE dimer, crystalline Pae SurEalpha is predominantly non-domain swapped. Comparative structural analyses of Tma and Pae SurE suggest conformationally variant regions, such as a hinge loop that may be involved in domain swapping. The putative SurE active site is highly conserved, and implies a model for SurE bound to a potential substrate, guanosine-5′-monophosphate (GMP). Pae SurEalpha has optimal acid phosphatase activity at temperatures above 90 degrees C, and is less specific than Tma SurE in terms of metal ion requirements. Substrate specificity also differs between Pae and Tma SurE, with a more specific recognition of purine nucleotides by the archaeal enzyme. Analyses of the sequences, phylogenetic distribution, and genomic organization of the SurE family reveal examples of genomes encoding multiple surE genes, and suggest that SurE homologs constitute a broad family of enzymes with phosphatase-like activities.

  • Mura C, Phillips M, Kozhukhovsky A, Eisenberg D. (2003). Structure and assembly of an augmented Sm-like archaeal protein 14-mer.Proc. Natl. Acad. Sci. U.S.A.. Apr 2003. 100(8):4539-44. [Abstract]

    To better understand the roles of Sm proteins in forming the cores of many RNA-processing ribonucleoproteins, we determined the crystal structure of an atypical Sm-like archaeal protein (SmAP3) in which the conserved Sm domain is augmented by a previously uncharacterized, mixed alpha/beta C-terminal domain. The structure reveals an unexpected SmAP3 14-mer that is perforated by a cylindrical pore and is bound to 14 cadmium (Cd(2+)) ions. Individual heptamers adopt either “apical” or “equatorial” conformations that chelate Cd(2+) differently. SmAP3 forms supraheptameric oligomers (SmAP3)(n = 7,14,28) in solution, and assembly of the asymmetric 14-mer is modulated by differential divalent cation-binding in apical and equatorial subunits. Phylogenetic and sequence analyses substantiate SmAP3s as a unique subset of SmAPs. These results distinguish SmAP3s from other Sm proteins and provide a model for the structure and properties of Sm proteins >100 residues in length, e.g., several human Sm proteins.

  • Mura C, Kozhukhovsky A, Gingery M, Phillips M, Eisenberg D. (2003). The oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs).Protein Sci.. Apr 2003. 12(4):832-47. [Abstract]

    Intron splicing is a prime example of the many types of RNA processing catalyzed by small nuclear ribonucleoprotein (snRNP) complexes. Sm proteins form the cores of most snRNPs, and thus to learn principles of snRNP assembly we characterized the oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs) from Pyrobaculum aerophilum (Pae) and Methanobacterium thermautotrophicum (Mth). Ultracentrifugation shows that Mth SmAP1 is exclusively heptameric in solution, whereas Pae SmAP1 forms either disulfide-bonded 14-mers or sub-heptameric states (depending on the redox potential). By electron microscopy, we show that Pae and Mth SmAP1 polymerize into bundles of well ordered fibers that probably form by head-to-tail stacking of heptamers. The crystallographic results reported here corroborate these findings by showing heptamers and 14-mers of both Mth and Pae SmAP1 in four new crystal forms. The 1.9 A-resolution structure of Mth SmAP1 bound to uridine-5′-monophosphate (UMP) reveals conserved ligand-binding sites. The likely RNA binding site in Mth agrees with that determined for Archaeoglobus fulgidus (Afu) SmAP1. Finally, we found that both Pae and Mth SmAP1 gel-shift negatively supercoiled DNA. These results distinguish SmAPs from eukaryotic Sm proteins and suggest that SmAPs have a generic single-stranded nucleic acid-binding activity.

  • Wang S, Eisenberg D. (2003). Crystal structures of a pantothenate synthetase from M. tuberculosis and its complexes with substrates and a reaction intermediate.Protein Sci.. May 2003. 12(5):1097-108. [Abstract]

    Pantothenate biosynthesis is essential for the virulence of Mycobacterium tuberculosis, and this pathway thus presents potential drug targets against tuberculosis. We determined the crystal structure of pantothenate synthetase (PS) from M. tuberculosis, and its complexes with AMPCPP, pantoate, and a reaction intermediate, pantoyl adenylate, with resolutions from 1.6 to 2 A. PS catalyzes the ATP-dependent condensation of pantoate and beta-alanine to form pantothenate. Its structure reveals a dimer, and each subunit has two domains with tight association between domains. The active-site cavity is on the N-terminal domain, partially covered by the C-terminal domain. One wall of the active site cavity is flexible, which allows the bulky AMPCPP to diffuse into the active site to nearly full occupancy when crystals are soaked in solutions containing AMPCPP. Crystal structures of the complexes with AMPCPP and pantoate indicate that the enzyme binds ATP and pantoate tightly in the active site, and brings the carboxyl oxygen of pantoate near the alpha-phosphorus atom of ATP for an in-line nucleophilic attack. When crystals were soaked with, or grown in the presence of, both ATP and pantoate, a reaction intermediate, pantoyl adenylate, is found in the active site. The flexible wall of the active site cavity becomes ordered when the intermediate is in the active site, thus protecting it from being hydrolyzed. Binding of beta-alanine can occur only after pantoyl adenylate is formed inside the active site cavity. The tight binding of the intermediate pantoyl adenylate suggests that nonreactive analogs of pantoyl adenylate may be inhibitors of the PS enzyme with high affinity and specificity.

  • Salwinski L, Eisenberg D. (2003). Computational methods of analysis of protein-protein interactions.Curr. Opin. Struct. Biol.. Jun 2003. 13(3):377-82. [Abstract]

    Computational methods play an important role at all stages of the process of determining protein-protein interactions. They are used to predict potential interactions, to validate the results of high-throughput interaction screens and to analyze the protein networks inferred from interaction databases.

  • Freedland SJ, Pantuck AJ, Paik SH, Zisman A, Graeber TG, Eisenberg D, McBride WH, Nguyen D, Tso CL, Belldegrun AS. (2003). Heterogeneity of molecular targets on clonal cancer lines derived from a novel hormone-refractory prostate cancer tumor system.Prostate. Jun 2003. 55(4):299-307. [Abstract]

    We recently described a new hormone refractory prostate cancer cell line, CL1, derived from LNCaP via in vitro androgen deprivation. To study gene expression during prostate cancer progression and to identify molecular targets for therapy, a pure clonal tumor system was generated.

  • Goulding CW, Perry LJ, Anderson D, Sawaya MR, Cascio D, Apostol MI, Chan S, Parseghian A, Wang SS, Wu Y, Cassano V, Gill HS, Eisenberg D. (2003). Structural genomics of Mycobacterium tuberculosis: a preliminary report of progress at UCLA.Biophys. Chem.. Sep 2003. 105(2-3):361-70. [Abstract]

    The growing list of fully sequenced genomes, combined with innovations in the fields of structural biology and bioinformatics, provides a synergy for the discovery of new drug targets. With this background, the TB Structural Genomics Consortium has been formed. This international consortium is comprised of laboratories from 31 universities and institutes in 13 countries. The goal of the consortium is to determine the structures of over 400 potential drug targets from the genome of Mycobacterium tuberculosis and analyze their structures in the context of functional information. We summarize the efforts of the UCLA consortium members. Potential drug targets were selected using a variety of bioinformatics methods and screened for certain physical and species-specific properties to yield a starting group of protein targets for structure determination. Target determination methods include protein phylogenetic profiles and Rosetta Stone methods, and the use of related biochemical pathways to select genes linked to essential prokaryotic genes. Criteria imposed on target selection included potential protein solubility, protein or domain size, and targets that lack homologs in eukaryotic organisms. In addition, some protein targets were chosen that are specific to M. tuberculosis, such as PE and PPE domains. Thus far, the UCLA group has cloned 263 targets, expressed 171 proteins and purified 40 proteins, which are currently in crystallization trials. Our efforts have yielded 13 crystals and eight structures. Seven structures are summarized here. Four of the structures are secreted proteins: antigen 85B; MPT 63, which is one of the three major secreted proteins of M. tuberculosis; a thioredoxin derivative Rv2878c; and potentially secreted glutamate synthetase. We also report the structures of three proteins that are potentially essential to the survival of M. tuberculosis: a protein involved in the folate biosynthetic pathway (Rv3607c); a protein involved in the biosynthesis of vitamin B5 (Rv3602c); and a pyrophosphatase, Rv2697c. Our approach to the M. tuberculosis structural genomics project will yield information for drug design and vaccine production against tuberculosis. In addition, this study will provide further insights into the mechanisms of mycobacterial pathogenesis.

  • Bleharski JR, Li H, Meinken C, Graeber TG, Ochoa MT, Yamamura M, Burdick A, Sarno EN, Wagner M, Röllinghoff M, Rea TH, Colonna M, Stenger S, Bloom BR, Eisenberg D, Modlin RL. (2003). Use of genetic profiling in leprosy to discriminate clinical forms of the disease.Science. Sep 2003. 301(5639):1527-30. [Abstract]

    Leprosy presents as a clinical and immunological spectrum of disease. With the use of gene expression profiling, we observed that a distinction in gene expression correlates with and accurately classifies the clinical form of the disease. Genes belonging to the leukocyte immunoglobulin-like receptor (LIR) family were significantly up-regulated in lesions of lepromatous patients suffering from the disseminated form of the infection. In functional studies, LIR-7 suppressed innate host defense mechanisms by shifting monocyte production from interleukin-12 toward interleukin-10 and by blocking antimicrobial activity triggered by Toll-like receptors. Gene expression profiles may be useful in defining clinical forms of disease and providing insights into the regulation of immune responses to pathogens.

  • Eisenberg D. (2003). The discovery of the alpha-helix and beta-sheet, the principal structural features of proteins.Proc. Natl. Acad. Sci. U.S.A.. Sep 2003. 100(20):11207-10. [Abstract]

    PNAS papers by Linus Pauling, Robert Corey, and Herman Branson in the spring of 1951 proposed the alpha-helix and the beta-sheet, now known to form the backbones of tens of thousands of proteins. They deduced these fundamental building blocks from properties of small molecules, known both from crystal structures and from Pauling’s resonance theory of chemical bonding that predicted planar peptide groups. Earlier attempts by others to build models for protein helices had failed both by including nonplanar peptides and by insisting on helices with an integral number of units per turn. In major respects, the Pauling-Corey-Branson models were astoundingly correct, including bond lengths that were not surpassed in accuracy for >40 years. However, they did not consider the hand of the helix or the possibility of bent sheets. They also proposed structures and functions that have not been found, including the gamma-helix.

  • Lee S, Eisenberg D. (2003). Seeded conversion of recombinant prion protein to a disulfide-bonded oligomer by a reduction-oxidation process.Nat. Struct. Biol.. Sep 2003. 10(9):725-30. [Abstract]

    The infectious form of prion protein, PrP(Sc), self-propagates by its conversion of the normal, cellular prion protein molecule PrP(C) to another PrP(Sc) molecule. It has not yet been demonstrated that recombinant prion protein can convert prion protein molecules from PrP(C) to PrP(Sc). Here we show that recombinant hamster prion protein is converted to a second form, PrP(RDX), by a redox process in vitro and that this PrP(RDX) form seeds the conversion of other PrP(C) molecules to the PrP(RDX) form. The converted form shows properties of oligomerization and seeded conversion that are characteristic of PrP(Sc). We also find that the oligomerization can be reversed in vitro. X-ray fiber diffraction suggests an amyloid-like structure for the oligomerized prion protein. A domain-swapping model involving intermolecular disulfide bonds can account for the stability and coexistence of two molecular forms of prion protein and the capacity of the second form for self-propagation.

  • Ivanova MI, Gingery M, Whitson LJ, Eisenberg D. (2003). Role of the C-terminal 28 residues of beta2-microglobulin in amyloid fibril formation.Biochemistry. Nov 2003. 42(46):13536-40. [Abstract]

    Beta2microglobulin (beta2m) is the major protein component of the fibrillar amyloid deposits isolated from patients diagnosed with dialysis-related amyloidosis (DRA). While investigating the molecular mechanism of amyloid fibril formation by beta2m, we found that the beta2m C-terminal peptide of 28 residues (cbeta2m) itself forms amyloid fibrils. When viewed by electron microscopy, cbeta2m aggregates appear as elongated unbranched fibers, the morphology typical for amyloids. Cbeta2m fibers stain with Congo red and show apple-green birefringence in polarized light, characteristic of amyloids. The observation that the beta2m C-terminal fragment readily forms amyloid fibrils implies that beta2m amyloid fibril formation proceeds via interactions of amyloid forming segments, which become exposed when the beta2m subunit is partially unfolded.

  • Strong M, Graeber TG, Beeby M, Pellegrini M, Thompson MJ, Yeates TO, Eisenberg D. (2003). Visualization and interpretation of protein networks in Mycobacterium tuberculosis based on hierarchical clustering of genome-wide functional linkage maps.Nucleic Acids Res.. Dec 2003. 31(24):7099-109. [Abstract]

    Genome-wide functional linkages among proteins in cellular complexes and metabolic pathways can be inferred from high throughput experimentation, such as DNA microarrays, or from bioinformatic analyses. Here we describe a method for the visualization and interpretation of genome-wide functional linkages inferred by the Rosetta Stone, Phylogenetic Profile, Operon and Conserved Gene Neighbor computational methods. This method involves the construction of a genome-wide functional linkage map, where each significant functional linkage between a pair of proteins is displayed on a two-dimensional scatter-plot, organized according to the order of genes along the chromosome. Subsequent hierarchical clustering of the map reveals clusters of genes with similar functional linkage profiles and facilitates the inference of protein function and the discovery of functionally linked gene clusters throughout the genome. We illustrate this method by applying it to the genome of the pathogenic bacterium Mycobacterium tuberculosis, assigning cellular functions to previously uncharacterized proteins involved in cell wall biosynthesis, signal transduction, chaperone activity, energy metabolism and polysaccharide biosynthesis.

  • Lee S, Sawaya MR, Eisenberg D. (2003). Structure of superoxide dismutase from Pyrobaculum aerophilum presents a challenging case in molecular replacement with multiple molecules, pseudo-symmetry and twinning.Acta Crystallogr. D Biol. Crystallogr.. Dec 2003. 59(Pt 12):2191-9. [Abstract]

    The crystal structure of superoxide dismutase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum was determined by molecular replacement at 1.8 A resolution. The structure determination was made especially challenging by the large number of molecules (24) in the asymmetric unit, the presence of a pseudo-crystallographic twofold operator close to a twinning operator and the inability to detect twinning by conventional means. Molecular replacement proceeded at low resolution in pseudo (apparent) space group P3(2)12 and was facilitated by examination of the self-rotation function and native Patterson map. Refinement, however, stalled at an R factor of 40% when high-resolution data were included. Expanding to the lower symmetry space group P3(2) decreased R (to 22%) and R(free) (to 26%), but not by as much as expected for the quality of data. Finally, despite the apparent lack of evidence from conventional twinning tests [i.e. plots of the second moment of I and N(Z) distributions], a twinning operator was included in the refinement, lowering R and R(free) to 16.2 and 21.7%, respectively. The early detection of twinning appears to have been masked by a deviation in the expected intensity distribution caused by the presence of non-crystallographic translational symmetry. These findings suggest the importance of testing twinning operators in cases where pseudo-translational symmetry can explain negative results from conventional twinning tests. The structure reveals a tetrameric assembly with 222 symmetry, similar to superoxide dismutase structures from other organisms. The current structural model represents the metal-free state of the enzyme.


  • Xenarios I, Salwínski L, Duan XJ, Higney P, Kim SM, Eisenberg D. (2002). DIP, the Database of Interacting Proteins: a research tool for studying cellular networks of protein interactions.Nucleic Acids Res.. Jan 2002. 30(1):303-5. [Abstract]

    The Database of Interacting Proteins (DIP: http://dip.doe-mbi.ucla.edu) is a database that documents experimentally determined protein-protein interactions. It provides the scientific community with an integrated set of tools for browsing and extracting information about protein interaction networks. As of September 2001, the DIP catalogs approximately 11 000 unique interactions among 5900 proteins from >80 organisms; the vast majority from yeast, Helicobacter pylori and human. Tools have been developed that allow users to analyze, visualize and integrate their own experimental data with the information about protein-protein interactions available in the DIP database.

  • Duan XJ, Xenarios I, Eisenberg D. (2002). Describing biological protein interactions in terms of protein states and state transitions: the LiveDIP database.Mol. Cell Proteomics. Feb 2002. 1(2):104-16. [Abstract]

    Biological protein-protein interactions differ from the more general class of physical interactions; in a biological interaction, both proteins must be in their proper states (e.g. covalently modified state, conformational state, cellular location state, etc.). Also in every biological interaction, one or both interacting molecules undergo a transition to a new state. This regulation of protein states through protein-protein interactions underlies many dynamic biological processes inside cells. Therefore, understanding biological interactions requires information on protein states. Toward this goal, DIP (the Database of Interacting Proteins) has been expanded to LiveDIP, which describes protein interactions by protein states and state transitions. This additional level of characterization permits a more complete picture of the protein-protein interaction networks and is crucial to an integrated understanding of genome-scale biology. The search tools provided by LiveDIP, Pathfinder, and Batch Search allow users to assemble biological pathways from all the protein-protein interactions collated from the scientific literature in LiveDIP. Tools have also been developed to integrate the protein-protein interaction networks of LiveDIP with large scale genomic data such as microarray data. An example of these tools applied to analyzing the pheromone response pathway in yeast suggests that the pathway functions in the context of a complex protein-protein interaction network. Seven of the eleven proteins involved in signal transduction are under negative or positive regulation of up to five other proteins through biological protein-protein interactions. During pheromone response, the mRNA expression levels of these signaling proteins exhibit different time course profiles. There is no simple correlation between changes in transcription levels and the signal intensity. This points to the importance of proteomic studies to understand how cells modulate and integrate signals. Integrating large scale, yeast two-hybrid data with mRNA expression data suggests biological interactions that may participate in pheromone response. These examples illustrate how LiveDIP provides data and tools for biological pathway discovery and pathway analysis.

  • Liu Y, Gotte G, Libonati M, Eisenberg D. (2002). Structures of the two 3D domain-swapped RNase A trimers.Protein Sci.. Feb 2002. 11(2):371-80. [Abstract]

    When concentrated in mildly acidic solutions, bovine pancreatic ribonuclease (RNase A) forms long-lived oligomers including two types of dimer, two types of trimer, and higher oligomers. In previous crystallographic work, we found that the major dimeric component forms by a swapping of the C-terminal beta-strands between the monomers, and that the minor dimeric component forms by swapping the N-terminal alpha-helices of the monomers. On the basis of these structures, we proposed that a linear RNase A trimer can form from a central molecule that simultaneously swaps its N-terminal helix with a second RNase A molecule and its C-terminal strand with a third molecule. Studies by dissociation are consistent with this model for the major trimeric component: the major trimer dissociates into both the major and the minor dimers, as well as monomers. In contrast, the minor trimer component dissociates into the monomer and the major dimer. This suggests that the minor trimer is cyclic, formed from three monomers that swap their C-terminal beta-strands into identical molecules. These conclusions are supported by cross-linking of lysyl residues, showing that the major trimer swaps its N-terminal helix, and the minor trimer does not. We verified by X-ray crystallography the proposed cyclic structure for the minor trimer, with swapping of the C-terminal beta-strands. This study thus expands the variety of domain-swapped oligomers by revealing the first example of a protein that can form both a linear and a cyclic domain-swapped oligomer. These structures permit interpretation of the enzymatic activities of the RNase A oligomers on double-stranded RNA.

  • Wang S, Mura C, Sawaya MR, Cascio D, Eisenberg D. (2002). Structure of a Nudix protein from Pyrobaculum aerophilum reveals a dimer with two intersubunit beta-sheets.Acta Crystallogr. D Biol. Crystallogr.. Apr 2002. 58(Pt 4):571-8. [Abstract]

    Nudix proteins, formerly called MutT homolog proteins, are a large family of proteins that play an important role in reducing the accumulation of potentially toxic compounds inside the cell. They hydrolyze a wide variety of substrates that are mainly composed of a nucleoside diphosphate linked to some other moiety X and thus are called Nudix hydrolases. Here, the crystal structure of a Nudix hydrolase from the hyperthermophilic archaeon Pyrobaculum aerophilum is reported. The structure was determined by the single-wavelength anomalous scattering method with data collected at the peak anomalous wavelength of an iridium-derivatized crystal. It reveals an extensive dimer interface, with each subunit contributing two strands to the beta-sheet of the other subunit. Individual subunits consist of a mixed highly twisted and curved beta-sheet of 11 beta-strands and two alpha-helices, forming an alpha-beta-alpha sandwich. The conserved Nudix box signature motif, which contains the essential catalytic residues, is located at the first alpha-helix and the beta-strand and loop preceding it. The unusually short connections between secondary-structural elements, together with the dimer form of the structure, are likely to contribute to the thermostability of the P. aerophilum Nudix protein.

  • Deane CM, Salwiński Ł, Xenarios I, Eisenberg D. (2002). Protein interactions: two methods for assessment of the reliability of high throughput observations.Mol. Cell Proteomics. May 2002. 1(5):349-56. [Abstract]

    High throughput methods for detecting protein interactions require assessment of their accuracy. We present two forms of computational assessment. The first method is the expression profile reliability (EPR) index. The EPR index estimates the biologically relevant fraction of protein interactions detected in a high throughput screen. It does so by comparing the RNA expression profiles for the proteins whose interactions are found in the screen with expression profiles for known interacting and non-interacting pairs of proteins. The second form of assessment is the paralogous verification method (PVM). This method judges an interaction likely if the putatively interacting pair has paralogs that also interact. In contrast to the EPR index, which evaluates datasets of interactions, PVM scores individual interactions. On a test set, PVM identifies correctly 40% of true interactions with a false positive rate of approximately 1%. EPR and PVM were applied to the Database of Interacting Proteins (DIP), a large and diverse collection of protein-protein interactions that contains over 8000 Saccharomyces cerevisiae pairwise protein interactions. Using these two methods, we estimate that approximately 50% of them are reliable, and with the aid of PVM we identify confidently 3003 of them. Web servers for both the PVM and EPR methods are available on the DIP website (dip.doe-mbi.ucla.edu/Services.cgi).

  • Kleiger G, Grothe R, Mallick P, Eisenberg D. (2002). GXXXG and AXXXA: common alpha-helical interaction motifs in proteins, particularly in extremophiles.Biochemistry. May 2002. 41(19):5990-7. [Abstract]

    The GXXXG motif is a frequently occurring sequence of residues that is known to favor helix-helix interactions in membrane proteins. Here we show that the GXXXG motif is also prevalent in soluble proteins whose structures have been determined. Some 152 proteins from a non-redundant PDB set contain at least one alpha-helix with the GXXXG motif, 41 +/- 9% more than expected if glycine residues were uniformly distributed in those alpha-helices. More than 50% of the GXXXG-containing alpha-helices participate in helix-helix interactions. In fact, 26 of those helix-helix interactions are structurally similar to the helix-helix interaction of the glycophorin A dimer, where two transmembrane helices associate to form a dimer stabilized by the GXXXG motif. As for the glycophorin A structure, we find backbone-to-backbone atomic contacts of the C alpha-H…O type in each of these 26 helix-helix interactions that display the stereochemical hallmarks of hydrogen bond formation. These glycophorin A-like helix-helix interactions are enriched in the general set of helix-helix interactions containing the GXXXG motif, suggesting that the inferred C alpha-H…O hydrogen bonds stabilize the helix-helix interactions. In addition to the GXXXG motif, some 808 proteins from the non-redundant PDB set contain at least one alpha-helix with the AXXXA motif (30 +/- 3% greater than expected). Both the GXXXG and AXXXA motifs occur frequently in predicted alpha-helices from 24 fully sequenced genomes. Occurrence of the AXXXA motif is enhanced to a greater extent in thermophiles than in mesophiles, suggesting that helical interaction based on the AXXXA motif may be a common mechanism of thermostability in protein structures. We conclude that the GXXXG sequence motif stabilizes helix-helix interactions in proteins, and that the AXXXA sequence motif also stabilizes the folded state of proteins.

  • Goulding CW, Sawaya MR, Parseghian A, Lim V, Eisenberg D, Missiakas D. (2002). Thiol-disulfide exchange in an immunoglobulin-like fold: structure of the N-terminal domain of DsbD.Biochemistry. Jun 2002. 41(22):6920-7. [Abstract]

    Escherichia coli DsbD transports electrons across the plasma membrane, a pathway that leads to the reduction of protein disulfide bonds. Three secreted thioredoxin-like factors, DsbC, DsbE, and DsbG, reduce protein disulfide bonds whereby an active site C-X-X-C motif is oxidized to generate a disulfide bond. DsbD catalyzes the reduction of the disulfide of DsbC, DsbE, and DsbG but not of the thioredoxin-like oxidant DsbA. The reduction of DsbC, DsbE, and DsbG occurs by transport of electrons from cytoplasmic thioredoxin to the C-terminal thioredoxin-like domain of DsbD (DsbD(C)). The N-terminal domain of DsbD, DsbD(N), acts as a versatile adaptor in electron transport and is capable of forming disulfides with oxidized DsbC, DsbE, or DsbG as well as with reduced DsbD(C). Isolated DsbD(N) is functional in electron transport in vitro. Crystallized DsbD(N) assumes an immunoglobulin-like fold that encompasses two active site cysteines, C103 and C109, forming a disulfide bond between beta-strands. The disulfide of DsbD(N) is shielded from the environment and capped by a phenylalanine (F70). A model is discussed whereby the immunoglobulin fold of DsbD(N) may provide for the discriminating interaction with thioredoxin-like factors, thereby triggering movement of the phenylalanine cap followed by disulfide rearrangement.

  • Liu Y, Eisenberg D. (2002). 3D domain swapping: as domains continue to swap.Protein Sci.. Jun 2002. 11(6):1285-99. [Abstract]

    Three-dimensional (3D) domain swapping creates a bond between two or more protein molecules as they exchange their identical domains. Since the term ‘3D domain swapping’ was first used to describe the dimeric structure of diphtheria toxin, the database of domain-swapped proteins has greatly expanded. Analyses of the now about 40 structurally characterized cases of domain-swapped proteins reveal that most swapped domains are at either the N or C terminus and that the swapped domains are diverse in their primary and secondary structures. In addition to tabulating domain-swapped proteins, we describe in detail several examples of 3D domain swapping which show the swapping of more than one domain in a protein, the structural evidence for 3D domain swapping in amyloid proteins, and the flexibility of hinge loops. We also discuss the physiological relevance of 3D domain swapping and a possible mechanism for 3D domain swapping. The present state of knowledge leads us to suggest that 3D domain swapping can occur under appropriate conditions in any protein with an unconstrained terminus. As domains continue to swap, this review attempts not only a summary of the known domain-swapped proteins, but also a framework for understanding future findings of 3D domain swapping.

  • Mallick P, Boutz DR, Eisenberg D, Yeates TO. (2002). Genomic evidence that the intracellular proteins of archaeal microbes contain disulfide bonds.Proc. Natl. Acad. Sci. U.S.A.. Jul 2002. 99(15):9679-84. [Abstract]

    Disulfide bonds have only rarely been found in intracellular proteins. That pattern is consistent with the chemically reducing environment inside the cells of well-studied organisms. However, recent experiments and new calculations based on genomic data of archaea provide striking contradictions to this pattern. Our results indicate that the intracellular proteins of certain hyperthermophilic archaea, especially the crenarchaea Pyrobaculum aerophilum and Aeropyrum pernix, are rich in disulfide bonds. This finding implicates disulfide bonding in stabilizing many thermostable proteins and points to novel chemical environments inside these microbes. These unexpected results illustrate the wealth of biochemical insights available from the growing reservoir of genomic data.

  • Gill HS, Pfluegl GM, Eisenberg D. (2002). Multicopy crystallographic refinement of a relaxed glutamine synthetase from Mycobacterium tuberculosis highlights flexible loops in the enzymatic mechanism and its regulation.Biochemistry. Aug 2002. 41(31):9863-72. [Abstract]

    The crystal structure of glutamine synthetase (GS) from Mycobacterium tuberculosis determined at 2.4 A resolution reveals citrate and AMP bound in the active site. The structure was refined with strict 24-fold noncrystallographic symmetry (NCS) constraints and has an R-factor of 22.7% and an R-free of 25.5%. Multicopy refinement using 10 atomic models and strict 24-fold NCS constraints further reduced the R-factor to 20.4% and the R-free to 23.2%. The multicopy model demonstrates the range of atomic displacements of catalytic and regulatory loops in glutamine synthesis, simulating loop motions. A comparison with loop positions in substrate complexes of GS from Salmonella typhimurium shows that the Asp50 and Glu327 loops close over the active site during catalysis. These loop closures are preceded by a conformational change of the Glu209 beta-strand upon metal ion or ATP binding that converts the enzyme from a relaxed to a taut state. We propose a model of the GS regulatory mechanism based on the loop motions in which adenylylation of the Tyr397 loop reverses the effect of metal ion binding, and regulates intermediate formation by preventing closure of the Glu327 loop.

  • Kleiger G, Eisenberg D. (2002). GXXXG and GXXXA motifs stabilize FAD and NAD(P)-binding Rossmann folds through C(alpha)-H… O hydrogen bonds and van der waals interactions.J. Mol. Biol.. Oct 2002. 323(1):69-76. [Abstract]

    Here we present evidence that domains in soluble proteins containing either the GXXXG or GXXXA motif are stabilized by the interaction of a beta-strand with the following alpha-helix. As an example, we characterized a beta-strand-helix interaction from the FAD or NAD(P)-binding Rossmann fold. The Rossmann fold is one of the three most highly represented folds in the Protein Data Bank (PDB). A subset of the proteins that adopt the Rossmann fold also bind to nucleotide cofactors such as FAD and NAD(P) and function as oxidoreductases. These Rossmann folds can often be identified by the short amino acid sequence motif, GX(1-2)GXXG. Here, we present evidence that in addition to this sequence motif, Rossmann folds that bind FAD and NAD(P) also typically contain either GXXXG or GXXXA motifs, where the first glycyl residue of these motifs and the third glycyl residue of the GX(1-2)GXXG motif are the same residue. These two motifs appear to stabilize the Rossmann fold: the first glycyl residue of either the GXXXG or GXXXA motif contacts the carbonyl oxygen atom from the first glycyl residue of the GX(1-2)GXXG motif consistent with the formation of a C(alpha)-H cdots, three dots, centered O hydrogen bond. In addition, both the glycyl and alanyl residues of the GXXXG or GXXXA motifs form van der Waals interactions with either a valine or isoleucine residue located either seven or eight residues further back along the polypeptide chain from the first glycine of the GXXXG or GXXXA motifs. Therefore, we combine both the GX(1-2)GXXG and GXXXG/A motifs into an extended motif, V/IXGX(1-2)GXXGXXXG/A, that is more strongly indicative than previously described motifs of Rossmann folds that bind FAD or NAD(P). The V/IXGX(1-2)GXXGXXXG/A motif can be used to search genomic sequence data and to annotate the function of proteins containing the motif as oxidoreductases, including proteins of previously unknown function.

  • Mallick P, Weiss R, Eisenberg D. (2002). The directional atomic solvation energy: an atom-based potential for the assignment of protein sequences to known folds.Proc. Natl. Acad. Sci. U.S.A.. Dec 2002. 99(25):16041-6. [Abstract]

    The Directional Atomic Solvation EnergY (DASEY) is an atom-based description of the environment of an amino acid position within a known 3D protein structure. The DASEY has been developed to align and score a probe amino acid sequence to a library of template protein structures for fold assignment. DASEY is computed by summing the atomic solvation parameters of atoms falling within a tetrahedral sector, or petal, extending 16 A along each of the four bond axes of each alpha-carbon atom of the protein. The DASEY discriminates between pairs of structurally equivalent positions and random pairs in protein structures sharing a fold but belonging to different superfamilies, unlike some previous descriptors of protein environments, such as buried area. Furthermore, the DASEY values have characteristic patterns of residue replacement, an essential feature of a successful fold assignment method. Benchmarking fold assignment with DASEY achieves coverage of 56% of sequences with 90% accuracy when probe sequences are matched to protein structural templates belonging to the same fold but to a different superfamily, an improvement of greater than 200% over a previous method.

  • Goulding CW, Parseghian A, Sawaya MR, Cascio D, Apostol MI, Gennaro ML, Eisenberg D. (2002). Crystal structure of a major secreted protein of Mycobacterium tuberculosis-MPT63 at 1.5-A resolution.Protein Sci.. Dec 2002. 11(12):2887-93. [Abstract]

    MPT63 is a small, major secreted protein of unknown function from Mycobacterium tuberculosis that has been shown to have immunogenic properties and has been implicated in virulence. A BLAST search identified that MPT63 has homologs only in other mycobacteria, and is therefore mycobacteria specific. As MPT63 is a secreted protein, mycobacteria specific, and implicated in virulence, MPT63 is an attractive drug target against the deadliest infectious disease, tuberculosis (TB). As part of the TB Structural Genomics Consortium, the X-ray crystal structure of MPT63 was determined to 1.5-Angstrom resolution with the hope of yielding functional information about MPT63. The structure of MPT63 is an antiparallel beta-sandwich immunoglobulin-like fold, with the unusual feature of the first beta-strand of the protein forming a parallel addition to the small antiparallel beta-sheet. MPT63 has weak structural similarity to many proteins with immunoglobulin folds, in particular, Homo sapiens beta2-adaptin, bovine arrestin, and Yersinia pseudotuberculosis invasin. Although the structure of MPT63 gives no conclusive evidence to its function, structural similarity suggests that MPT63 could be involved in cell-host interactions to facilitate endocytosis/phagocytosis.


  • Xenarios I, Fernandez E, Salwinski L, Duan XJ, Thompson MJ, Marcotte EM, Eisenberg D. (2001). DIP: The Database of Interacting Proteins: 2001 update.Nucleic Acids Res.. Jan 2001. 29(1):239-41. [Abstract]

    The Database of Interacting Proteins (DIP; http://dip.doe-mbi.ucla. edu) is a database that documents experimentally determined protein-protein interactions. Since January 2000 the number of protein-protein interactions in DIP has nearly tripled to 3472 and the number of proteins to 2659. New interactive tools have been developed to aid in the visualization, navigation and study of networks of protein interactions.

  • Gill HS, Eisenberg D. (2001). The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition.Biochemistry. Feb 2001. 40(7):1903-12. [Abstract]

    Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS). The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 A resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods. The structure suggests a noncovalent, dead-end mechanism of inhibition. Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme. One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327. The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket. The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin. The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands. The inhibition of GS by methionine sulfoximine can be explained by the same mechanism. These models of inhibited GS further illuminate its catalytic mechanism.

  • Balbirnie M, Grothe R, Eisenberg DS. (2001). An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated beta-sheet structure for amyloid.Proc. Natl. Acad. Sci. U.S.A.. Feb 2001. 98(5):2375-80. [Abstract]

    X-ray diffraction and other biophysical tools reveal features of the atomic structure of an amyloid-like crystal. Sup35, a prion-like protein in yeast, forms fibrillar amyloid assemblies intrinsic to its prion function. We have identified a polar peptide from the N-terminal prion-determining domain of Sup35 that exhibits the amyloid properties of full-length Sup35, including cooperative kinetics of aggregation, fibril formation, binding of the dye Congo red, and the characteristic cross-beta x-ray diffraction pattern. Microcrystals of this peptide also share the principal properties of the fibrillar amyloid, including a highly stable, beta-sheet-rich structure and the binding of Congo red. The x-ray powder pattern of the microcrystals, extending to 0.9-A resolution, yields the unit cell dimensions of the well-ordered structure. These dimensions restrict possible atomic models of this amyloid-like structure and demonstrate that it forms packed, parallel-stranded beta-sheets. The unusually high density of the crystals shows that the packed beta-sheets are dehydrated, despite the polar character of the side chains. These results suggest that amyloid is a highly intermolecularly bonded, dehydrated array of densely packed beta-sheets. This dry beta-sheet could form as Sup35 partially unfolds to expose the peptide, permitting it to hydrogen-bond to the same peptide of other Sup35 molecules. The implication is that amyloid-forming units may be short segments of proteins, exposed for interactions by partial unfolding.

  • Ogihara NL, Ghirlanda G, Bryson JW, Gingery M, DeGrado WF, Eisenberg D. (2001). Design of three-dimensional domain-swapped dimers and fibrous oligomers.Proc. Natl. Acad. Sci. U.S.A.. Feb 2001. 98(4):1404-9. [Abstract]

    Three-dimensional (3D) domain-swapped proteins are intermolecularly folded analogs of monomeric proteins; both are stabilized by the identical interactions, but the individual domains interact intramolecularly in monomeric proteins, whereas they form intermolecular interactions in 3D domain-swapped structures. The structures and conditions of formation of several domain-swapped dimers and trimers are known, but the formation of higher order 3D domain-swapped oligomers has been less thoroughly studied. Here we contrast the structural consequences of domain swapping from two designed three-helix bundles: one with an up-down-up topology, and the other with an up-down-down topology. The up-down-up topology gives rise to a domain-swapped dimer whose structure has been determined to 1.5 A resolution by x-ray crystallography. In contrast, the domain-swapped protein with an up-down-down topology forms fibrils as shown by electron microscopy and dynamic light scattering. This demonstrates that design principles can predict the oligomeric state of 3D domain-swapped molecules, which should aid in the design of domain-swapped proteins and biomaterials.

  • Anderson DH, Harth G, Horwitz MA, Eisenberg D. (2001). An interfacial mechanism and a class of inhibitors inferred from two crystal structures of the Mycobacterium tuberculosis 30 kDa major secretory protein (Antigen 85B), a mycolyl transferase.J. Mol. Biol.. Mar 2001. 307(2):671-81. [Abstract]

    The Mycobacterium tuberculosis 30 kDa major secretory protein (antigen 85B) is the most abundant protein exported by M. tuberculosis, as well as a potent immunoprotective antigen and a leading drug target. A mycolyl transferase of 285 residues, it is closely related to two other mycolyl transferases, each of molecular mass 32 kDa: antigen 85A and antigen 85C. All three catalyze transfer of the fatty acid mycolate from one trehalose monomycolate to another, resulting in trehalose dimycolate and free trehalose, thus helping to build the bacterial cell wall. We have determined two crystal structures of M. tuberculosis antigen 85B (ag85B), initially by molecular replacement using antigen 85C as a probe. The apo ag85B model is refined against 1.8 A data, to an R-factor of 0.196 (R(free) is 0.276), and includes all residues except the N-terminal Phe. The active site immobilizes a molecule of the cryoprotectant 2-methyl-2,4-pentanediol. Crystal growth with addition of trehalose resulted in a second ag85B crystal structure (1.9 A resolution; R-factor is 0.195; R(free) is 0.285). Trehalose binds in two sites at opposite ends of the active-site cleft. In our proposed mechanism model, the trehalose at the active site Ser126 represents the trehalose liberated by temporary esterification of Ser126, while the other trehalose represents the incoming trehalose monomycolate just prior to swinging over to the first trehalose site to displace the mycolate from its serine ester. Our proposed interfacial mechanism minimizes aqueous exposure of the apolar mycolates. Based on the trehalose-bound structure, we suggest a new class of antituberculous drugs, made by connecting two trehalose molecules by an amphipathic linker.

  • Liu Y, Gotte G, Libonati M, Eisenberg D. (2001). A domain-swapped RNase A dimer with implications for amyloid formation.Nat. Struct. Biol.. Mar 2001. 8(3):211-4. [Abstract]

    Bovine pancreatic ribonuclease (RNase A) forms two types of dimers (a major and a minor component) upon concentration in mild acid. These two dimers exhibit different biophysical and biochemical properties. Earlier we reported that the minor dimer forms by swapping its N-terminal alpha-helix with that of an identical molecule. Here we find that the major dimer forms by swapping its C-terminal beta-strand, thus revealing the first example of three-dimensional (3D) domain swapping taking place in different parts of the same protein. This feature permits RNase A to form tightly bonded higher oligomers. The hinge loop of the major dimer, connecting the swapped beta-strand to the protein core, resembles a short segment of the polar zipper proposed by Perutz and suggests a model for aggregate formation by 3D domain swapping with a polar zipper.

  • Marcotte EM, Xenarios I, Eisenberg D. (2001). Mining literature for protein-protein interactions.Bioinformatics. Apr 2001. 17(4):359-63. [Abstract]

    MOTIVATION: A central problem in bioinformatics is how to capture information from the vast current scientific literature in a form suitable for analysis by computer. We address the special case of information on protein-protein interactions, and show that the frequencies of words in Medline abstracts can be used to determine whether or not a given paper discusses protein-protein interactions. For those papers determined to discuss this topic, the relevant information can be captured for the Database of Interacting PROTEINS: Furthermore, suitable gene annotations can also be captured. RESULTS: Our Bayesian approach scores Medline abstracts for probability of discussing the topic of interest according to the frequencies of discriminating words found in the abstract. More than 80 discriminating words (e.g. complex, interaction, two-hybrid) were determined from a training set of 260 Medline abstracts corresponding to previously validated entries in the Database of Interacting Proteins. Using these words and a log likelihood scoring function, approximately 2000 Medline abstracts were identified as describing interactions between yeast proteins. This approach now forms the basis for the rapid expansion of the Database of Interacting Proteins.

  • Landgraf R, Xenarios I, Eisenberg D. (2001). Three-dimensional cluster analysis identifies interfaces and functional residue clusters in proteins.J. Mol. Biol.. Apr 2001. 307(5):1487-502. [Abstract]

    Three-dimensional cluster analysis offers a method for the prediction of functional residue clusters in proteins. This method requires a representative structure and a multiple sequence alignment as input data. Individual residues are represented in terms of regional alignments that reflect both their structural environment and their evolutionary variation, as defined by the alignment of homologous sequences. From the overall (global) and the residue-specific (regional) alignments, we calculate the global and regional similarity matrices, containing scores for all pairwise sequence comparisons in the respective alignments. Comparing the matrices yields two scores for each residue. The regional conservation score (C(R)(x)) defines the conservation of each residue x and its neighbors in 3D space relative to the protein as a whole. The similarity deviation score (S(x)) detects residue clusters with sequence similarities that deviate from the similarities suggested by the full-length sequences. We evaluated 3D cluster analysis on a set of 35 families of proteins with available cocrystal structures, showing small ligand interfaces, nucleic acid interfaces and two types of protein-protein interfaces (transient and stable). We present two examples in detail: fructose-1,6-bisphosphate aldolase and the mitogen-activated protein kinase ERK2. We found that the regional conservation score (C(R)(x)) identifies functional residue clusters better than a scoring scheme that does not take 3D information into account. C(R)(x) is particularly useful for the prediction of poorly conserved, transient protein-protein interfaces. Many of the proteins studied contained residue clusters with elevated similarity deviation scores. These residue clusters correlate with specificity-conferring regions: 3D cluster analysis therefore represents an easily applied method for the prediction of functionally relevant spatial clusters of residues in proteins.

  • Mura C, Cascio D, Sawaya MR, Eisenberg DS. (2001). The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core.Proc. Natl. Acad. Sci. U.S.A.. May 2001. 98(10):5532-7. [Abstract]

    Sm proteins form the core of small nuclear ribonucleoprotein particles (snRNPs), making them key components of several mRNA-processing assemblies, including the spliceosome. We report the 1.75-A crystal structure of SmAP, an Sm-like archaeal protein that forms a heptameric ring perforated by a cationic pore. In addition to providing direct evidence for such an assembly in eukaryotic snRNPs, this structure (i) shows that SmAP homodimers are structurally similar to human Sm heterodimers, (ii) supports a gene duplication model of Sm protein evolution, and (iii) offers a model of SmAP bound to single-stranded RNA (ssRNA) that explains Sm binding-site specificity. The pronounced electrostatic asymmetry of the SmAP surface imparts directionality to putative SmAP-RNA interactions.

  • Xenarios I, Eisenberg D. (2001). Protein interaction databases.Curr. Opin. Biotechnol.. Aug 2001. 12(4):334-9. [Abstract]

    Life depends on the interaction of proteins. The availability of the complete human genome sequence has highlighted the need for a tool to analyse protein interactions and several databases have been compiled for this purpose. These databases document, categorize, and analyze interacting proteins and the cellular functions of the interactions.

  • Dym O, Eisenberg D. (2001). Sequence-structure analysis of FAD-containing proteins.Protein Sci.. Sep 2001. 10(9):1712-28. [Abstract]

    We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein ‘pharmacophore’ exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.

  • Graeber TG, Eisenberg D. (2001). Bioinformatic identification of potential autocrine signaling loops in cancers from gene expression profiles.Nat. Genet.. Nov 2001. 29(3):295-300. [Abstract]

    Many biological signaling pathways involve autocrine ligand-receptor loops; misregulation of these signaling loops can contribute to cancer phenotypes. Here we present an algorithm for detecting such loops from gene expression profiles. Our method is based on the hypothesis that for some autocrine pathways, the ligand and receptor are regulated by coupled mechanisms at the level of transcription, and thus ligand-receptor pairs comprising such a loop should have correlated mRNA expression. Using our database of experimentally known ligand-receptor signaling partners, we found examples of ligand-receptor pairs with significantly correlated expression in five cancer-based gene expression datasets. The correlated ligand-receptor pairs we identified are consistent with known autocrine signaling events in cancer cells. In addition, our algorithm predicts new autocrine signaling loops that can be verified experimentally. Chemokines were commonly members of these potential autocrine pathways. Our analysis also revealed ligand-receptor pairs with expression patterns that may indicate cellular mechanisms for preventing autocrine signaling.

  • Singer E, Landgraf R, Horan T, Slamon D, Eisenberg D. (2001). Identification of a heregulin binding site in HER3 extracellular domain.J. Biol. Chem.. Nov 2001. 276(47):44266-74. [Abstract]

    HER3 (also known as c-Erb-b3) is a type I receptor tyrosine kinase similar in sequence to the epidermal growth factor (EGF) receptor. The extracellular segment of this transmembrane receptor contains four domains. Domains I and II are similar in sequence to domains III and IV, respectively, and domains II and IV are cysteine-rich. We show that the EGF-like domain of heregulin (hrg) binds to domains I and II of HER3, in contrast to the EGF receptor, for which prior studies have shown that a construct consisting of domains III and portions of domain IV binds EGF. Next, we identified a putative hrg binding site by limited proteolysis of the recombinant extracellular domains of HER3 (HER3-ECD(I-IV)) in both the presence and absence of hrg. In the absence of hrg, HER3-ECD(I-IV) is cleaved after position Tyr(50), near the beginning of domain I. Binding of hrg to HER3-ECD(I-IV) fully protects position Tyr(50) from proteolysis. To confirm that domain I contains a hrg binding site, we expressed domains I and II (HER3-ECD(I-II)) and find that it binds hrg with 68 nm affinity. These data suggest that domains I and II of HER3-ECD(I-IV) act as a functional unit in folding and binding of hrg. Thus, our biochemical findings reinforce the structural hypothesis of others that HER3-ECD(I-IV) is similar to the insulin-like growth factor-1 receptor (IGF-1R), as follows: 1) The protected cleavage site in HER3-ECD(I-IV) corresponds to a binding footprint in domain I of IGF-1R; 2) HER3-ECD(I-II) binds hrg with a 68 nm dissociation constant, supporting the hypothesis that domain I is involved in ligand binding; and 3) the large accessible surface area (1749 A) of domain L1 of IGF-1R that is buried by domain S1, as well as the presence of conserved contacts in this interface of type 1 RTKs, suggests that domains L1 and S1 of IGF-1R function as a unit as observed for HER3-ECD(I-II). Our results are consistent with the proposal that HER3 has a structure similar to IGF-1R and binds ligand at a site in corresponding domains.

  • Kleiger G, Perry J, Eisenberg D. (2001). 3D structure and significance of the GPhiXXG helix packing motif in tetramers of the E1beta subunit of pyruvate dehydrogenase from the archeon Pyrobaculum aerophilum.Biochemistry. Dec 2001. 40(48):14484-92. [Abstract]

    As part of a structural genomics project, we have determined the 2.0 A structure of the E1beta subunit of pyruvate dehydrogenase from Pyrobaculum aerophilum (PA), a thermophilic archaeon. The overall fold of E1beta from PA is closely similar to the previously determined E1beta structures from humans (HU) and P. putida (PP). However, unlike the HU and PP structures, the PA structure was determined in the absence of its partner subunit, E1alpha. Significant structural rearrangements occur in E1beta when its E1alpha partner is absent, including rearrangement of several secondary structure elements such as helix C. Helix C is buried by E1alpha in the HU and PP structures, but makes crystal contacts in the PA structure that lead to an apparent beta(4) tetramer. Static light scattering and sedimentation velocity data are consistent with the formation of PA E1beta tetramers in solution. The interaction of helix C with its symmetry-related counterpart stabilizes the tetrameric interface, where two glycine residues on the same face of one helix create a packing surface for the other helix. This GPhiXXG helix-helix interaction motif has previously been found in interacting transmembrane helices, and is found here at the E1alpha-E1beta interface for both the HU and PP alpha(2)beta(2) tetramers. As a case study in structural genomics, this work illustrates that comparative analysis of protein structures can identify the structural significance of a sequence motif.

  • Salwinski L, Eisenberg D. (2001). Motif-based fold assignment.Protein Sci.. Dec 2001. 10(12):2460-9. [Abstract]

    Conventional fold recognition techniques rely mainly on the analysis of the entire sequence of a protein. We present an MBA method to improve performance of any conventional sequence-based fold assignment. The method uses sequence motifs, such as those defined in the Prosite database, and the SwissProt annotation of the fold library. When combined with a simple SDP method, the coverage of MBA is comparable to the results obtained with PSI-BLAST. However, the set of the MBA predictions is significantly different from that of PSI-BLAST, leading to a 40% increase of the coverage for the combined MBA/PSI-BLAST method. The MBA approach can be easily adopted to include the results of sequence-independent function prediction methods and alternative motif and annotation databases. The method is available through the web server localized at http://www.doe-mbi.ucla.edu/mba.


  • Diehl DL, Eisenberg D. (2000). Complementary and alternative medicine (CAM): epidemiology and implications for research.Prog. Brain Res.. 2000. 122:445-55. [Abstract]
  • Xenarios I, Rice DW, Salwinski L, Baron MK, Marcotte EM, Eisenberg D. (2000). DIP: the database of interacting proteins.Nucleic Acids Res.. Jan 2000. 28(1):289-91. [Abstract]

    The Database of Interacting Proteins (DIP; http://dip.doe-mbi.ucla.edu) is a database that documents experimentally determined protein-protein interactions. This database is intended to provide the scientific community with a comprehensive and integrated tool for browsing and efficiently extracting information about protein interactions and interaction networks in biological processes. Beyond cataloging details of protein-protein interactions, the DIP is useful for understanding protein function and protein-protein relationships, studying the properties of networks of interacting proteins, benchmarking predictions of protein-protein interactions, and studying the evolution of protein-protein interactions.

  • Eisenberg D, Gill HS, Pfluegl GM, Rotstein SH. (2000). Structure-function relationships of glutamine synthetases.Biochim. Biophys. Acta. Mar 2000. 1477(1-2):122-45. [Abstract]

    As a highly regulated enzyme at the core of nitrogen metabolism, glutamine synthetase has been studied intensively. We review structural and functional studies of both bacterial and eukaryotic glutamine synthetases, with emphasis on enzymatic inhibitors.

  • Mallick P, Goodwill KE, Fitz-Gibbon S, Miller JH, Eisenberg D. (2000). Selecting protein targets for structural genomics of Pyrobaculum aerophilum: validating automated fold assignment methods by using binary hypothesis testing.Proc. Natl. Acad. Sci. U.S.A.. Mar 2000. 97(6):2450-5. [Abstract]

    Three-dimensional protein folds were assigned to all ORFs of the recently sequenced genome of the hyperthermophilic archaeon Pyrobaculum aerophilum. Binary hypothesis testing was used to estimate a confidence level for each assignment. A separate test was conducted to assign a probability for whether each sequence has a novel fold-i.e., one that is not yet represented in the experimental database of known structures. Of the 2,130 predicted nontransmembrane proteins in this organism, 916 matched a fold at a cumulative 90% confidence level, and 245 could be assigned at a 99% confidence level. Likewise, 286 proteins were predicted to have a previously unobserved fold with a 90% confidence level, and 14 at a 99% confidence level. These statistically based tools are combined with homology searches against the Online Mendelian Inheritance in Man (OMIM) human genetics database and other protein databases for the selection of attractive targets for crystallographic or NMR structure determination. Results of these studies have been collated and placed at http://www.doe-mbi.ucla.edu/people/parag/P A_HOME/, the University of California, Los Angeles-Department of Energy Pyrobaculum aerophilum web site.

  • Ruth L, Eisenberg D, Neufeld EF. (2000). alpha-L-iduronidase forms semi-crystalline spherulites with amyloid-like properties.Acta Crystallogr. D Biol. Crystallogr.. Apr 2000. 56(Pt 4):524-8. [Abstract]

    While seeking conditions for single crystals of human alpha-L-iduronidase, solutions were discovered (pH 3.0-8.5 containing calcium or zinc salts) that transform soluble alpha-L-iduronidase to a solid aggregate. This aggregate is a spherulite of semi-crystalline protein. The X-ray diffraction pattern and ability to bind Congo red characterize the alpha-L-iduronidase spherulite as ‘amyloid-like’, in that it displays two of the characteristics of amyloidogenic proteins. In addition, alpha-L-iduronidase also interacts with heparin, as do some amyloid-forming proteins.

  • Eisenberg D, Marcotte EM, Xenarios I, Yeates TO. (2000). Protein function in the post-genomic era.Nature. Jun 2000. 405(6788):823-6. [Abstract]

    Faced with the avalanche of genomic sequences and data on messenger RNA expression, biological scientists are confronting a frightening prospect: piles of information but only flakes of knowledge. How can the thousands of sequences being determined and deposited, and the thousands of expression profiles being generated by the new array methods, be synthesized into useful knowledge? What form will this knowledge take? These are questions being addressed by scientists in the field known as ‘functional genomics’.

  • Kleiger G, Beamer LJ, Grothe R, Mallick P, Eisenberg D. (2000). The 1.7 A crystal structure of BPI: a study of how two dissimilar amino acid sequences can adopt the same fold.J. Mol. Biol.. Jun 2000. 299(4):1019-34. [Abstract]

    We have extended the resolution of the crystal structure of human bactericidal/permeability-increasing protein (BPI) to 1.7 A. BPI has two domains with the same fold, but with little sequence similarity. To understand the similarity in structure of the two domains, we compare the corresponding residue positions in the two domains by the method of 3D-1D profiles. A 3D-1D profile is a string formed by assigning each position in the 3D structure to one of 18 environment classes. The environment classes are defined by the local secondary structure, the area of the residue which is buried from solvent, and the fraction of the area buried by polar atoms. A structural alignment between the two BPI domains was used to compare the 3D-1D environments of structurally equivalent positions. Greater than 31% of the aligned positions have conserved 3D-1D environments, but only 13% have conserved residue identities. Analysis of the 3D-1D environmentally conserved positions helps to identify pairs of residues likely to be important in conserving the fold, regardless of the residue similarity. We find examples of 3D-1D environmentally conserved positions with dissimilar residues which nevertheless play similar structural roles. To generalize our findings, we analyzed four other proteins with similar structures yet dissimilar sequences. Together, these examples show that aligned pairs of dissimilar residues often share similar structural roles, stabilizing dissimilar sequences in the same fold.

  • Landgraf R, Eisenberg D. (2000). Heregulin reverses the oligomerization of HER3.Biochemistry. Jul 2000. 39(29):8503-11. [Abstract]

    We analyzed the propensity of the HER3 receptor and its extracellular domain (ECD) to undergo ligand-independent self-association. The HER3-ECD, purified from Drosophila S2 cells, binds the EGF-like domain of heregulin (hrg) with a K(d) of 1.9 nM as measured by surface plasmon resonance (SPR) studies. In a gel shift assay, the HER3-ECD self-associates into a uniform, slowly migrating species in a concentration-dependent manner, starting at concentrations of <10 nM. In contrast to the HER3-ECD, the ECD from the related HER2 receptor does not oligomerize under the same conditions. The direct interaction of HER3-ECDs was also demonstrated by pull-down assays and SPR measurements under physiological salt conditions. This self-association of the HER3-ECD was reversed by the addition of hrg but not by EGF. The apparent equilibrium dissociation constant for the HER3-ECD self-association is 15 nM, based on SPR measurements. In this analysis, hrg blocks HER3-ECD self-association, and the addition of hrg during the dissociation phase resulted in an accelerated off rate. This finding suggests that hrg can bind to and disrupt preexisting HER3-ECD oligomers. Full-length HER3 likewise exhibited self-association. Under conditions where co-immunoprecipitation and cross-linking of HER2 and HER3 were stimulated by hrg, HER3 self-association and cross-linking were disrupted by hrg. The implication is that the self-association of HER3-ECD favors the formation of catalytically inactive complexes of the HER3 receptor. Binding of hrg releases HER3 which may then form signaling-competent HER3-HER2 heterodimers.

  • Dym O, Pratt EA, Ho C, Eisenberg D. (2000). The crystal structure of D-lactate dehydrogenase, a peripheral membrane respiratory enzyme.Proc. Natl. Acad. Sci. U.S.A.. Aug 2000. 97(17):9413-8. [Abstract]

    d-Lactate dehydrogenase (d-LDH) of Escherichia coli is a peripheral membrane respiratory enzyme involved in electron transfer, located on the cytoplasmic side of the inner membrane. d-LDH catalyzes the oxidation of d-lactate to pyruvate, which is coupled to transmembrane transport of amino acids and sugars. Here we describe the crystal structure at 1.9 A resolution of the three domains of d-LDH: the flavin adenine dinucleotide (FAD)-binding domain, the cap domain, and the membrane-binding domain. The FAD-binding domain contains the site of d-lactate reduction by a noncovalently bound FAD cofactor and has an overall fold similar to other members of a recently discovered FAD-containing family of proteins. This structural similarity extends to the cap domain as well. The most prominent difference between d-LDH and the other members of the FAD-containing family is the membrane-binding domain, which is either absent in some of these proteins or differs significantly. The d-LDH membrane-binding domain presents an electropositive surface with six Arg and five Lys residues, which presumably interacts with the negatively charged phospholipid head groups of the membrane. Thus, d-LDH appears to bind the membrane through electrostatic rather than hydrophobic forces.

  • Marcotte EM, Xenarios I, van Der Bliek AM, Eisenberg D. (2000). Localizing proteins in the cell from their phylogenetic profiles.Proc. Natl. Acad. Sci. U.S.A.. Oct 2000. 97(22):12115-20. [Abstract]

    We introduce a computational method for identifying subcellular locations of proteins from the phylogenetic distribution of the homologs of organellar proteins. This method is based on the observation that proteins localized to a given organelle by experiments tend to share a characteristic phylogenetic distribution of their homologs-a phylogenetic profile. Therefore any other protein can be localized by its phylogenetic profile. Application of this method to mitochondrial proteins reveals that nucleus-encoded proteins previously known to be destined for mitochondria fall into three groups: prokaryote-derived, eukaryote-derived, and organism-specific (i.e., found only in the organism under study). Prokaryote-derived mitochondrial proteins can be identified effectively by their phylogenetic profiles. In the yeast Saccharomyces cerevisiae, 361 nucleus-encoded mitochondrial proteins can be identified at 50% accuracy with 58% coverage. From these values and the proportion of conserved mitochondrial genes, it can be inferred that approximately 630 genes, or 10% of the nuclear genome, is devoted to mitochondrial function. In the worm Caenorhabditis elegans, we estimate that there are approximately 660 nucleus-encoded mitochondrial genes, or 4% of its genome, with approximately 400 of these genes contributed from the prokaryotic mitochondrial ancestor. The large fraction of organism-specific and eukaryote-derived genes suggests that mitochondria perform specialized roles absent from prokaryotic mitochondrial ancestors. We observe measurably distinct phylogenetic profiles among proteins from different subcellular compartments, allowing the general use of prokaryotic genomes in learning features of eukaryotic proteins.

  • Steere B, Eisenberg D. (2000). Characterization of high-order diphtheria toxin oligomers.Biochemistry. Dec 2000. 39(51):15901-9. [Abstract]

    In 3D domain swapping, a domain of a protein breaks its noncovalent bonds with the protein core and its place is taken by the identical domain of another molecule, creating a strongly bound dimer or higher order oligomer. For some proteins, including diphtheria toxin, 3D domain swapping may affect protein function. To explore the molecular basis of 3D domain swapping in a well-characterized protein system, domain-swapped oligomers of diphtheria toxin were produced by freezing and thawing under a variety conditions, including in various salts and buffers, and at various temperatures. Reaction yields were followed by high-performance size-exclusion chromatography. The traditional low pH pulse produced by freeze-thawing in mixed sodium phosphate buffer induces the oligomerization of DT, but the addition of alkali chloride salts was found to increase the yield in the order of Li(+) > Na(+) > K(+). Unexpectedly, oligomers also formed when DT was frozen and thawed in the presence of 1 M LiCl alone. Slower freezing and thawing of the mixture led to the production of more and larger oligomers. DT oligomers were also produced by exposure to acidic buffers, and were found by electron microscopy to adopt both linear and cyclized forms in a wide distribution of sizes. Upon the basis of these results, the model for the production of DT oligomers by freezing and thawing was expanded to include a salt-mediated pathway. We present a mechanism for the formation of high-order DT oligomers by acidification that takes into account domain swapping and hydrophobic interactions.


  • Marcotte EM, Eisenberg D. (1999). Chicken prion tandem repeats form a stable, protease-resistant domain.Biochemistry. Jan 1999. 38(2):667-76. [Abstract]

    Prion-linked diseases, such as mad cow disease, scrapie, and the human genetic disorder Creutzfeldt-Jakob disease, are fatal neurodegenerative diseases correlated with changes in the secondary structure of neural prion protein. We expressed recombinant chicken prion protein in Escherichia coli and purified the protein to homogeneity. Circular dichroism spectra of the 26 kDa recombinant protein closely resemble those of prion protein purified directly from healthy hamster brain. The chicken prion protein exists as a soluble, monodisperse monomer but can be forced to multimerize following lyophilization and resuspension. We analyzed the chicken prion protein domain structure by proteolysis and show that, unlike the mammalian homologues, the chicken prion protein N-terminal tandem amino acid repeats form a stable, protease-resistant domain. This domain probably represents a physiologically functional unit. As tested by both mass spectrometry and circular dichroism, the mature chicken prion protein does not bind copper, unlike synthetic peptides from the chicken prion N-terminus, suggesting that binding copper is not the physiological activity of the chicken prion. However, copper strongly destabilizes the prion protein and depresses the melting temperature by 30 degreesC, presumably by binding to the unfolded form of the prion protein. The chicken prion N-terminus may have evolved to fold without a cofactor, unlike mammalian prion proteins, whose N-termini are disordered without cofactors such as copper present. Chicken prion offers an alternative to intractable mammalian prions for structural studies of the amino-terminal domain.

  • Hart PJ, Balbirnie MM, Ogihara NL, Nersissian AM, Weiss MS, Valentine JS, Eisenberg D. (1999). A structure-based mechanism for copper-zinc superoxide dismutase.Biochemistry. Feb 1999. 38(7):2167-78. [Abstract]

    A reaction cycle is proposed for the mechanism of copper-zinc superoxide dismutase (CuZnSOD) that involves inner sphere electron transfer from superoxide to Cu(II) in one portion of the cycle and outer sphere electron transfer from Cu(I) to superoxide in the other portion of the cycle. This mechanism is based on three yeast CuZnSOD structures determined by X-ray crystallography together with many other observations. The new structures reported here are (1) wild type under 15 atm of oxygen pressure, (2) wild type in the presence of azide, and (3) the His48Cys mutant. Final R-values for the three structures are respectively 20.0%, 17.3%, and 20.9%. Comparison of these three new structures to the wild-type yeast Cu(I)ZnSOD model, which has a broken imidazolate bridge, reveals the following: (i) The protein backbones (the “SOD rack”) remain essentially unchanged. (ii) A pressure of 15 atm of oxygen causes a displacement of the copper ion 0.37 A from its Cu(I) position in the trigonal plane formed by His46, His48, and His120. The displacement is perpendicular to this plane and toward the NE2 atom of His63 and is accompanied by elongated copper electron density in the direction of the displacement suggestive of two copper positions in the crystal. The copper geometry remains three coordinate, but the His48-Cu bond distance increases by 0.18 A. (iii) Azide binding also causes a displacement of the copper toward His63 such that it moves 1.28 A from the wild-type Cu(I) position, but unlike the effect of 15 atm of oxygen, there is no two-state character. The geometry becomes five-coordinate square pyramidal, and the His63 imidazolate bridge re-forms. The His48-Cu distance increases by 0.70 A, suggesting that His48 becomes an axial ligand. (iv) The His63 imidazole ring tilts upon 15 atm of oxygen treatment and azide binding. Its NE2 atom moves toward the trigonal plane by 0.28 and 0.66 A, respectively, in these structures. (v) The replacement of His48 by Cys, which does not bind copper, results in a five-coordinate square pyramidal, bridge-intact copper geometry with a novel chloride ligand. Combining results from these and other CuZnSOD crystal structures, we offer the outlines of a structure-based cyclic mechanism.

  • Pellegrini M, Marcotte EM, Thompson MJ, Eisenberg D, Yeates TO. (1999). Assigning protein functions by comparative genome analysis: protein phylogenetic profiles.Proc. Natl. Acad. Sci. U.S.A.. Apr 1999. 96(8):4285-8. [Abstract]

    Determining protein functions from genomic sequences is a central goal of bioinformatics. We present a method based on the assumption that proteins that function together in a pathway or structural complex are likely to evolve in a correlated fashion. During evolution, all such functionally linked proteins tend to be either preserved or eliminated in a new species. We describe this property of correlated evolution by characterizing each protein by its phylogenetic profile, a string that encodes the presence or absence of a protein in every known genome. We show that proteins having matching or similar profiles strongly tend to be functionally linked. This method of phylogenetic profiling allows us to predict the function of uncharacterized proteins.

  • Gill HS, Pfluegl GM, Eisenberg D. (1999). Preliminary crystallographic studies on glutamine synthetase from Mycobacterium tuberculosis.Acta Crystallogr. D Biol. Crystallogr.. Apr 1999. 55(Pt 4):865-8. [Abstract]

    The etiologic agent of tuberculosis, Mycobacterium tuberculosis, has been shown to secrete the enzyme glutamine synthetase (TB-GS) which is apparently essential for infection. Four crystal forms of a recombinant TB-GS were grown. The one chosen for synchrotron X–ray data collection belongs to space group P212121 with unit-cell dimensions 208 x 258 x 274 A, yielding 2.4 A resolution data. A Matthews number of 2.89 A3 Da-1 is found, corresponding to 24 subunits of molecular mass 1300 kDa in the asymmetric unit. From earlier work, the structure of Salmonella typhimurium GS, which is 51% identical in sequence to TB-GS, is known to be dodecameric with 622 symmetry. Self-rotation calculations on the TB-GS X-ray data reveal only one set of sixfold and twofold axes of symmetry. A Patterson map calculated from the native X-ray data confirms that there are two dodecamers in the asymmetric unit, having both their sixfold and twofold axes parallel to one another.

  • Marcotte EM, Pellegrini M, Ng HL, Rice DW, Yeates TO, Eisenberg D. (1999). Detecting protein function and protein-protein interactions from genome sequences.Science. Jul 1999. 285(5428):751-3. [Abstract]

    A computational method is proposed for inferring protein interactions from genome sequences on the basis of the observation that some pairs of interacting proteins have homologs in another organism fused into a single protein chain. Searching sequences from many genomes revealed 6809 such putative protein-protein interactions in Escherichia coli and 45,502 in yeast. Many members of these pairs were confirmed as functionally related; computational filtering further enriches for interactions. Some proteins have links to several other proteins; these coupled links appear to represent functional interactions such as complexes or pathways. Experimentally confirmed interacting pairs are documented in a Database of Interacting Proteins.

  • Patterson WR, Anderson DH, DeGrado WF, Cascio D, Eisenberg D. (1999). Centrosymmetric bilayers in the 0.75 A resolution structure of a designed alpha-helical peptide, D,L-Alpha-1.Protein Sci.. Jul 1999. 8(7):1410-22. [Abstract]

    We report the 0.75 A crystal structure of a racemic mixture of the 12-residue designed peptide “Alpha-1” (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1bar. The unit cell contains two L-alpha-helices and two D-alpha-helices. The columnar-sheet bilayer motif seen in L-Alpha-1 is maintained in the D,L-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the L-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion.

  • Thompson MJ, Eisenberg D. (1999). Transproteomic evidence of a loop-deletion mechanism for enhancing protein thermostability.J. Mol. Biol.. Jul 1999. 290(2):595-604. [Abstract]

    Understanding the molecular determinants of protein thermostability is of theoretical and practical importance. While numerous determinants have been suggested, no molecular feature has been judged of paramount importance, with the possible exception of ion-pair networks. The difficulty in identifying the main determinants may have been the limited structural information available on the thermostable proteins. Recently the complete genomes for mesophilic, thermophilic and hyperthermophilic organisms have been sequenced, vastly improving the potential for uncovering general trends in sequence and structure evolution related to thermostability and, thus, for isolating the more important determinants. From a comparative analysis of 20 complete genomes, we find a trend towards shortened thermophilic proteins relative to their mesophilic homologs. Moreover, sequence alignments to proteins of known structure indicate that thermophilic sequences are more likely than their mesophilic homologs to have deletions in exposed loop regions. The new genomes offer enough comparable sequences to compute meaningful statistics that point to loop deletion as a general evolutionary strategy for increasing thermostability.

  • Eisenberg D. (1999). How chaperones protect virgin proteins.Science. Aug 1999. 285(5430):1021-2. [Abstract]
  • Eisenberg D. (1999). Glories of protein structureStructure. Oct 1999. 7(10):R241-2. [Abstract]
  • Marcotte EM, Pellegrini M, Yeates TO, Eisenberg D. (1999). A census of protein repeats.J. Mol. Biol.. Oct 1999. 293(1):151-60. [Abstract]

    In this study, we analyzed all known protein sequences for repeating amino acid segments. Although duplicated sequence segments occur in 14 % of all proteins, eukaryotic proteins are three times more likely to have internal repeats than prokaryotic proteins. After clustering the repetitive sequence segments into families, we find repeats from eukaryotic proteins have little similarity with prokaryotic repeats, suggesting most repeats arose after the prokaryotic and eukaryotic lineages diverged. Consequently, protein classes with the highest incidence of repetitive sequences perform functions unique to eukaryotes. The frequency distribution of the repeating units shows only weak length dependence, implicating recombination rather than duplex melting or DNA hairpin formation as the limiting mechanism underlying repeat formation. The mechanism favors additional repeats once an initial duplication has been incorporated. Finally, we show that repetitive sequences are favored that contain small and relatively water-soluble residues. We propose that error-prone repeat expansion allows repetitive proteins to evolve more quickly than non-repeat-containing proteins.

  • Landgraf R, Fischer D, Eisenberg D. (1999). Analysis of heregulin symmetry by weighted evolutionary tracing.Protein Eng.. Nov 1999. 12(11):943-51. [Abstract]

    Heregulins are members of the protein family of EGF-like growth and differentiation factors. The primary cell-surface targets of heregulins are heterodimers of the EGF-receptor homolog HER2 with either HER3 or HER4. We used a weighted evolutionary trace analysis to identify structural features that distinguish the EGF-like domain (hrg) of heregulins from other members of the EGF family. In this analysis, each amino acid sequence is weighted according to its uniqueness and the variability in each position is assigned by an amino acid substitution matrix. Conserved residues in heregulin that are variable in other EGF-like domains are considered possible specificity-conferring residues. This analysis identifies two clusters of residues at the foot of the boot-shaped hrg domain. The residues in one cluster are recruited from the N-terminus; those in the other are from the ohm-loop region and show a weak sequence similarity to the N-terminal residues at the opposite side of the boot. The remaining residues with high conservation scores distribute themselves into these two distinct surfaces on hrg. This pseudo-twofold symmetry and the presence of two distinct interfaces may reflect the preference of hrg for heterodimeric versus homodimeric HER complexes.

  • Marcotte EM, Pellegrini M, Thompson MJ, Yeates TO, Eisenberg D. (1999). A combined algorithm for genome-wide prediction of protein function.Nature. Nov 1999. 402(6757):83-6. [Abstract]

    The availability of over 20 fully sequenced genomes has driven the development of new methods to find protein function and interactions. Here we group proteins by correlated evolution, correlated messenger RNA expression patterns and patterns of domain fusion to determine functional relationships among the 6,217 proteins of the yeast Saccharomyces cerevisiae. Using these methods, we discover over 93,000 pairwise links between functionally related yeast proteins. Links between characterized and uncharacterized proteins allow a general function to be assigned to more than half of the 2,557 previously uncharacterized yeast proteins. Examples of functional links are given for a protein family of previously unknown function, a protein whose human homologues are implicated in colon cancer and the yeast prion Sup35.


  • Liu Y, Hart PJ, Schlunegger MP, Eisenberg D. (1998). The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-A resolution.Proc. Natl. Acad. Sci. U.S.A.. Mar 1998. 95(7):3437-42. [Abstract]

    The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-A resolution. The dimer is 3D domain-swapped. The N-terminal helix (residues 1-15) of each subunit is swapped into the major domain (residues 23-124) of the other subunit. The dimer of bull seminal ribonuclease (BS-RNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of approximately 160 degrees. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the “closed interface” but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the “open interface.” The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.

  • Hart PJ, Liu H, Pellegrini M, Nersissian AM, Gralla EB, Valentine JS, Eisenberg D. (1998). Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis.Protein Sci.. Mar 1998. 7(3):545-55. [Abstract]

    The X-ray crystal structure of a human copper/zinc superoxide dismutase mutant (G37R CuZnSOD) found in some patients with the inherited form of Lou Gehrig’s disease (FALS) has been determined to 1.9 angstroms resolution. The two SOD subunits have distinct environments in the crystal and are different in structure at their copper binding sites. One subunit (subunit[intact]) shows a four-coordinate ligand geometry of the copper ion, whereas the other subunit (subunit[broken]) shows a three-coordinate geometry of the copper ion. Also, subunit(intact) displays higher atomic displacement parameters for backbone atoms ((B) = 30 +/- 10 angstroms2) than subunit(broken) ((B) = 24 +/- 11 angstroms2). This structure is the first CuZnSOD to show large differences between the two subunits. Factors that may contribute to these differences are discussed and a possible link of a looser structure to FALS is suggested.

  • Landgraf R, Pegram M, Slamon DJ, Eisenberg D. (1998). Cytotoxicity and specificity of directed toxins composed of diphtheria toxin and the EGF-like domain of heregulin beta1.Biochemistry. Mar 1998. 37(9):3220-8. [Abstract]

    As a step in the design of directed toxins, aimed at cells that overexpress HER receptors, particularly breast carcinoma cells, we studied the properties of a chimera of diphtheria toxin (DT) and heregulin beta1. The EGF-like growth hormone heregulin is a ligand for the HER3 and HER4 receptors and their heterodimers with HER2. The 60-residue EGF-like domain (hrg) of heregulin elicits a biological response and binds to these receptors primarily through its N terminus. We tested a fusion protein in which hrg replaces the C-terminal receptor-binding domain of DT (DT(389)hrg) and an alternative design in which this domain is fused to the N terminus of DT(389). Of those two constructs, the N-terminal fusion was not active as a directed toxin but elicited a growth response. The C-terminal fusion of hrg to DT(389) yielded a functional toxin and showed cell line specific cytotoxicity that is consistent with heregulin specificity. The binding of hrg to its cognate receptor is not impaired as shown by receptor activation, direct binding, and competition with free hrg. Cytotoxicity is dependent on high-affinity binding of DT(389)hrg to HER3 and HER4 receptors and is not mediated by HER2 overexpression alone. For those cell lines exhibiting high-affinity binding sites, the level of cytotoxicity correlates with the rate of internalization. Thus DT(389)hrg chimeras offer a possible avenue toward directed toxins against cells that overexpress HER receptors.

  • Beamer LJ, Carroll SF, Eisenberg D. (1998). The BPI/LBP family of proteins: a structural analysis of conserved regions.Protein Sci.. Apr 1998. 7(4):906-14. [Abstract]

    Two related mammalian proteins, bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP), share high-affinity binding to lipopolysaccharide (LPS), a glycolipid found in the outer membrane of gram-negative bacteria. The recently determined crystal structure of human BPI permits a structure/function analysis, presented here, of the conserved regions of these two proteins sequences. In the seven known sequences of BPI and LBP, 102 residues are completely conserved and may be classified in terms of location, side-chain chemistry, and interactions with other residues. We find that the most highly conserved regions lie at the interfaces between the tertiary structural elements that help create two apolar lipid-binding pockets. Most of the conserved polar and charged residues appear to be involved in inter-residue interactions such as H-bonding. However, in both BPI and LBP a subset of conserved residues with positive charge (lysines 42, 48, 92, 95, and 99 of BPI) have no apparent structural role. These residues cluster at the tip of the NH2-terminal domain, and several coincide with residues known to affect LPS binding; thus, it seems likely that these residues make electrostatic interactions with negatively charged groups of LPS. Overall differences in charge and electrostatic potential between BPI and LBP suggest that BPI’s bactericidal activity is related to the high positive charge of its NH2-terminal domain. A model of human LBP derived from the BPI structure provides a rational basis for future experiments, such as site-directed mutagenesis and inhibitor design.

  • Peer Bork, David Eisenberg. (1998). Sequences and topology Deriving biological knowledge from genomic sequences Curr. Opin. Struct. Biol.. June 1998. 8(3): 331-332 [Abstract]

    No abstract is available for this article.

  • Beamer LJ, Fischer D, Eisenberg D. (1998). Detecting distant relatives of mammalian LPS-binding and lipid transport proteins.Protein Sci.. Jul 1998. 7(7):1643-6. [Abstract]

    In mammals, a family of four lipid binding proteins has been previously defined that includes two lipopolysaccharide binding proteins and two lipid transfer proteins. The first member of this family to have its three-dimensional structure determined is bactericidal/permeability-increasing protein (BPI). Using both the sequence and structure of BPI, along with recently developed sequence-sequence and sequence-structure similarity search methods, we have identified 13 distant members of the family in a diverse set of eukaryotes, including rat, chicken, Caenorhabditis elegans, and Biomphalaria galbrata. Although the sequence similarity between these 13 new members and any of the 4 original members of the BPI family is well below the “twilight zone,” their high sequence-structure compatibility with BPI indicates they are likely to share its fold. These findings broaden the BPI family to include a member found in retina and brain, and suggest that a primitive member may have contained only one of the two similar domains of BPI.


  • Rice DW, Fischer D, Weiss R, Eisenberg D. (1997). Fold assignments for amino acid sequences of the CASP2 experiment.Proteins. 1997. Suppl 1:113-22. [Abstract]

    New and newly extended methods for fold assignment were tested for their abilities to assign folds to amino acid target sequences of unknown three-dimensional structure. These target sequences, released through the CASP2 experiment, are not obviously related to any sequence of known three-dimensional (3D) structure. We assigned 3D folds to target sequences and filed these predictions with CASP2 before their 3D structures were released. The methods tested were (1) Environmental 3D profiles of Bowie and colleagues [Bowie, J.U., Luthy, R., Eisenberg, D. Science 253:164-170, 1991]; (2) A variation of this is termed Directional Profiles; (3) The H3P2 five-dimensional sequence-structure substitution matrix of Rice and Eisenberg [Rice, D., Eisenberg, D.J. Mol. Biol. 267:1026-1037, 1997]; and (4) The Sequence Derived Property methods of Fischer and Eisenberg [Fischer, D., Eisenberg, D. Prot. Sci. 5:947-955, 1996]. When the 3D structures of the sequences were released, 17 of our predictions were evaluated. Of these 17, we assigned high probabilities to 11, of which 9 were correct. Five of these correct predictions were of known 3D structures similar to the targets and four of these were of new folds. The evaluation demonstrated that our methods were effective in assigning the proper fold to more than half of the CASP2 targets with known folds (5/9) and also were able to detect half of the sequences that corresponded to no known folds (4/8). Even when the correct fold is assigned to a sequence, proper alignment of the sequence to the structure remains a challenge. Our methods were able to produce accurate alignments (< 1.2 mean residue shift error from the structural alignment) for four of the targets, including the particularly difficult alignment (only 7% residue identity in the structurally aligned regions) of the ferrochelatase sequence to the fold of a periplasmic binding protein.

  • Eisenberg D, Lüthy R, Bowie JU. (1997). VERIFY3D: assessment of protein models with three-dimensional profiles.Meth. Enzymol.. 1997. 277:396-404. [Abstract]
  • Schlunegger MP, Bennett MJ, Eisenberg D. (1997). Oligomer formation by 3D domain swapping: a model for protein assembly and misassembly.Adv. Protein Chem.. 1997. 50:61-122. [Abstract]
  • Bell CE, Eisenberg D. (1997). Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide.Adv. Exp. Med. Biol.. 1997. 419:35-43. [Abstract]

    The crystal structure of diphtheria toxin (DT) in complex with nicotinamide adenine dinucleotide (NAD) has been determined by x-ray crystallography to 2.3 A resolution. NAD binds to a cleft on the surface of the catalytic (C) domain of DT, interacting closely with the side chains of Tyr54, Tyr65, His21, Thr23, and Glu 48. The carboxylate group of Glu148 of Dt lies approximately 4 A from the scissile, N-glycosidic bound of NAD, suggesting a possible catalytic role for Glu148 in stabilizing a positively charged oxocarbonium intermediate. Residues 39-46 of the active-site loop of the C-domain become disordered upon NAD-binding, suggesting a potential role for these residues in binding to elongation facor-2 (EF-2). Structural alignments of the DT-NAD complex with the structures of other ADP-ribosylating toxins suggest how NAD may bind to these other enzymes.

  • Bell CE, Eisenberg D. (1997). Crystal structure of nucleotide-free diphtheria toxin.Biochemistry. Jan 1997. 36(3):481-8. [Abstract]

    The crystal structure of diphtheria toxin (DT) in the absence of nucleotide (nucleotide-free DT) has been determined at 2.3 A resolution to a crystallographic R factor and free R factor of 18.2 and 28.2%, respectively. A comparison of this structure to the previously determined structures of DT in complex with adenyly(3′-5′)uridine monophosphate (ApUp) and DT in complex with nicotinamide adenine dinucleotide (NAD) reveals that there are no significant movements of the two subdomains of the catalytic (C) domain associated with dinucleotide binding. The side chains of six residues within the active-site cleft, including Tyr65, Pro38, Tyr27, Thr23, Glu148, and Tyr54, show movements of up to 3 A upon dinucleotide binding. In the structure of nucleotide-free DT, the active-site loop residues 39-47 of the C domain are well ordered and extend over the active-site cleft in approximately the same position as in the structure of DT in complex with ApUp. This is in contrast to the structure of the DT-NAD complex, in which the active-site loop is disordered. On the basis of a comparison of the nucleotide-free and NAD-bound DT structures, we suggest that the interaction of NAD with Pro38 and also possibly Tyr54 and Trp153 could disrupt the network of hydrogen bonds that stabilizes the position of the active-site loop over the active-site cleft, allowing this loop to become disordered. This may be an important step in binding of the C domain of DT to its substrate, elongation factor-2.

  • Ogihara NL, Weiss MS, Degrado WF, Eisenberg D. (1997). The crystal structure of the designed trimeric coiled coil coil-VaLd: implications for engineering crystals and supramolecular assemblies.Protein Sci.. Jan 1997. 6(1):80-8. [Abstract]

    The three-dimensional structure of the 29-residue designed coiled coil having the amino acid sequence acetyl-E VEALEKK VAALESK VQALEKK VEALEHG-amide has been determined and refined to a crystallographic R-factor of 21.4% for all data from 10-A to 2.1-A resolution. This molecule is called coil-VaLd because it contains valine in the a heptad positions and leucine in the d heptad positions. In the trigonal crystal, three molecules, related by a crystallographic threefold axis, form a parallel three-helix bundle. The bundles are stacked head-to-tail to form a continuous coiled coil along the c-direction of the crystal. The contacts among the three helices within the coiled coil are mainly hydrophobic: four layers of valine residues alternate with four layers of leucine residues to form the core of the bundle. In contrast, mostly hydrophilic contacts mediate the interaction between trimers: here a total of two direct protein–protein hydrogen bonds are found. Based on the structure, we propose a scheme for designing crystals of peptides containing continuous two-, three-, and four-stranded coiled coils.

  • Eisenberg D. (1997). Into the black of night.Nat. Struct. Biol.. Feb 1997. 4(2):95-7. [Abstract]
  • Rice DW, Eisenberg D. (1997). A 3D-1D substitution matrix for protein fold recognition that includes predicted secondary structure of the sequence.J. Mol. Biol.. Apr 1997. 267(4):1026-38. [Abstract]

    In protein fold recognition, a probe amino acid sequence is compared to a library of representative folds of known structure to identify a structural homolog. In cases where the probe and its homolog have clear sequence similarity, traditional residue substitution matrices have been used to predict the structural similarity. In cases where the probe is sequentially distant from its homolog, we have developed a (7 x 3 x 2 x 7 x 3) 3D-1D substitution matrix (called H3P2), calculated from a database of 119 structural pairs. Members of each pair share a similar fold, but have sequence identity less than 30%. Each probe sequence position is defined by one of seven residue classes and three secondary structure classes. Each homologous fold position is defined by one of seven residue classes, three secondary structure classes, and two burial classes. Thus the matrix is five-dimensional and contains 7 x 3 x 2 x 7 x 3 = 882 elements or 3D-1D scores. The first step in assigning a probe sequence to its homologous fold is the prediction of the three-state (helix, strand, coil) secondary structure of the probe; here we use the profile based neural network prediction of secondary structure (PHD) program. Then a dynamic programming algorithm uses the H3P2 matrix to align the probe sequence with structures in a representative fold library. To test the effectiveness of the H3P2 matrix a challenging, fold class diverse, and cross-validated benchmark assessment is used to compare the H3P2 matrix to the GONNET, PAM250, BLOSUM62 and a secondary structure only substitution matrix. For distantly related sequences the H3P2 matrix detects more homologous structures at higher reliabilities than do these other substitution matrices, based on sensitivity versus specificity plots (or SENS-SPEC plots). The added efficacy of the H3P2 matrix arises from its information on the statistical preferences for various sequence-structure environment combinations from very distantly related proteins. It introduces the predicted secondary structure information from a sequence into fold recognition in a statistical way that normalizes the inherent correlations between residue type, secondary structure and solvent accessibility.

  • Beamer LJ, Carroll SF, Eisenberg D. (1997). Crystal structure of human BPI and two bound phospholipids at 2.4 angstrom resolution.Science. Jun 1997. 276(5320):1861-4. [Abstract]

    Bactericidal/permeability-increasing protein (BPI), a potent antimicrobial protein of 456 residues, binds to and neutralizes lipopolysaccharides from the outer membrane of Gram-negative bacteria. At a resolution of 2.4 angstroms, the crystal structure of human BPI shows a boomerang-shaped molecule formed by two similar domains. Two apolar pockets on the concave surface of the boomerang each bind a molecule of phosphatidylcholine, primarily by interacting with their acyl chains; this suggests that the pockets may also bind the acyl chains of lipopolysaccharide. As a model for the related plasma lipid transfer proteins, BPI illuminates a mechanism of lipid transfer for this protein family.

  • Anderson DH, Weiss MS, Eisenberg D. (1997). Charges, hydrogen bonds, and correlated motions in the 1 A resolution refined structure of the mating pheromone Er-1 from Euplotes raikovi.J. Mol. Biol.. Oct 1997. 273(2):479-500. [Abstract]

    A detailed description is given of the structure of the small protein mating pheromone Er-1 at atomic resolution. Emphasis is placed on the locations of charges and hydrogen bonds. The model includes all the protein atoms, anisotropic displacement parameters, four disordered side chains, 22 water molecules, a disordered ethanol, and “riding” hydrogen atoms. Analysis of the model revealed that this dense crystal is perfused by hydrogen-bonding networks of solvent and protein atoms. The termini of helices are capped by hydrogen bonding to solvent and protein atoms, and to symmetry-related molecules. An examination of the valencies and charges of the hydrogen-bonding groups suggests that three of the “water” molecules capping the C termini of two helices, and one other, may instead be NH4 ions. Water molecules mediate all but one of the interhelical hydrogen bonds, and many of the lattice interactions. Regions of the molecule where the atomic vibrations deviate from isotropy are identified. There is almost no overall libration of the molecule allowed by the packing, but the side-chains vibrate relative to the backbone. Four side-chains display alternate conformations. Indirect evidence is presented that the switches between their conformations are correlated and driven by protonation of acidic side-chains. These structural features are discussed in the context of function and stability. Equipped with the analysis of the model, we review the course and results of the refinement of the model against 1 A X-ray diffraction data to a crystallographic R-factor of 12.92%.

  • Fischer D, Eisenberg D. (1997). Assigning folds to the proteins encoded by the genome of Mycoplasma genitalium.Proc. Natl. Acad. Sci. U.S.A.. Oct 1997. 94(22):11929-34. [Abstract]

    A crucial step in exploiting the information inherent in genome sequences is to assign to each protein sequence its three-dimensional fold and biological function. Here we describe fold assignment for the proteins encoded by the small genome of Mycoplasma genitalium. The assignment was carried out by our computer server (http://www.doe-mbi.ucla.edu/people/frsvr/ frsvr. html), which assigns folds to amino acid sequences by comparing sequence-derived predictions with known structures. Of the total of 468 protein ORFs, 103 (22%) can be assigned a known protein fold with high confidence, as cross-validated with tests on known structures. Of these sequences, 75 (16%) show enough sequence similarity to proteins of known structure that they can also be detected by traditional sequence-sequence comparison methods. That is, the difference of 28 sequences (6%) are assignable by the sequence-structure method of the server but not by current sequence-sequence methods. Of the remaining 78% of sequences in the genome, 18% belong to membrane proteins and the remaining 60% cannot be assigned either because these sequences correspond to no presently known fold or because of insensitivity of the method. At the current rate of determination of new folds by x-ray and NMR methods, extrapolation suggests that folds will be assigned to most soluble proteins in the next decade.

  • Bell CE, Yeates TO, Eisenberg D. (1997). Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: a comparison with NAD bound to the oxidoreductase enzymes.Protein Sci.. Oct 1997. 6(10):2084-96. [Abstract]

    The conformation of NAD bound to diphtheria toxin (DT), an ADP-ribosylating enzyme, has been compared to the conformations of NAD(P) bound to 23 distinct NAD(P)-binding oxidoreductase enzymes, whose structures are available in the Brookhaven Protein Data Bank. For the oxidoreductase enzymes, NAD(P) functions as a cofactor in electron transfer, whereas for DT, NAD is a labile substrate in which the N-glycosidic bond between the nicotinamide ring and the N-ribose is cleaved. All NAD(P) conformations were compared by (1) visual inspection of superimposed molecules, (2) RMSD of atomic positions, (3) principal component analysis, and (4) analysis of torsion angles and other conformational parameters. Whereas the majority of oxidoreductase-bound NAD(P) conformations are found to be similar, the conformation of NAD bound to DT is found to be unusual. Distinctive features of the conformation of NAD bound to DT that may be relevant to DT’s function as an ADP-ribosylating enzyme include (1) an unusually short distance between the PN and N1N atoms, reflecting a highly folded conformation for the nicotinamide mononucleotide (NMN) portion of NAD, and (2) a torsion angle chi N approximately 0 degree about the scissile N-glycosidic bond, placing the nicotinamide ring outside of the preferred anti and syn orientations. In NAD bound to DT, the highly folded NMN conformation and torsion angle chi N approximately 0 degree could contribute to catalysis, possibly by orienting the C1’N atom of NAD for nucleophilic attack, or by placing strain on the N-glycosidic bond, which is cleaved by DT. The unusual overall conformation of NAD bound to DT is likely to reflect the structure of DT, which is unusual among NAD(P)-binding enzymes. In DT, the NAD binding site is formed at the junction of two antiparallel beta-sheets. In contrast, although the 24 oxidoreductase enzymes belong to at least six different structural classes, almost all of them bind NAD(P) at the C-terminal end of a parallel beta-sheet. The structural alignments and principal component analysis show that enzymes of the same structural class bind to particularly similar conformations of NAD(P), with few exceptions. The conformation of NAD bound to DT superimposes closely with that of an NAD analogue bound to Pseudomonas exotoxin A, an ADP-ribosylating toxin that is structurally homologous to DT. This suggests that all of the ADP-ribosylating enzymes that are structurally homologous to DT and ETA will bind a highly similar conformation of NAD.

  • Bell CE, Poon PH, Schumaker VN, Eisenberg D. (1997). Oligomerization of a 45 kilodalton fragment of diphtheria toxin at pH 5.0 to a molecule of 20-24 subunits.Biochemistry. Dec 1997. 36(49):15201-7. [Abstract]

    Diphtheria toxin (DT) is a 58 kDa protein, secreted by lysogenic strains of Corynebacterium diphtheriae, that causes the disease diphtheria in humans. The catalytic (C) domain of DT kills host cells by gaining entry into the cytoplasm and inhibiting protein synthesis. The translocation of the C domain across the endosomal membrane and into the cytoplasm of a host cell is mediated by the translocation (T) domain of DT. This process is triggered by acidification from pH approximately 7 to pH approximately 5 within the endosome. Here we show that crm45 (cross-reacting material of 45 kDa), a 45 kDa deletion mutant of DT which contains the C and T domains but lacks the C-terminal receptor-binding (R) domain, undergoes a transition from a monomer to a large oligomer upon acidification from pH 7.0 to pH 5.0. Dynamic light scattering analysis of crm45 at pH 5.0 results in a polydispersity value of only 8-17%, suggesting that the oligomer is uniformly sized. Using analytical ultracentrifugation, measurements of the sedimentation rate and diffusion coefficient of crm45 at pH 5.0 result in a molecular mass determination of 890 +/- 40 kDa (20 +/- 1 subunits) for the oligomer. Equilibrium sedimentation data on crm45 at pH 5.0 are best fit by a single species with a mass of 1000 +/- 50 kDa (24 +/- 1 subunits). These results reveal the pH-dependent formation of a uniformly sized, 20-24 subunit oligomer of the C and T domains of DT, in solution. Because the oligomer of crm45 forms at the pH of the acidified endosome, it could be relevant to the translocation of the C domain of DT across the endosomal membrane and into the cytoplasm of host cells. The possible relevance of this oligomer of crm45 to the membrane translocation of the C domain of DT correlates with earlier kinetic studies of DT intoxication of Vero cells, which inferred the transfer of approximately 20 C domains of DT to the cytoplasm of host cells, in a single event.


  • Fischer D, Elofsson A, Rice D, Eisenberg D. (1996). Assessing the performance of fold recognition methods by means of a comprehensive benchmark.Pac Symp Biocomput. 1996. :300-18. [Abstract]

    Recently there has been an explosion of methods for fold recognition. These methods seek to align a protein sequence to a three-dimensional structure and measure the compatibility of the sequence to the structure. In this work, we present a benchmark to assess the performance of such methods. The benchmark consists of a set of protein sequences matched by superposition to known structures. This set covers a wide range of protein families, and includes matching proteins with insignificant sequence similarity. To demonstrate the usefulness of this benchmark, we apply it here to compare different fold-recognition methods developed through the years in our group as well as several sequence-sequence substitution matrices. The results show that “global-local” alignments are superior to either local or global alignments. The most effective sequence-sequence matching matrix is the Gonnet table. The best performance overall is obtained by a method which combines the 3D-1D profiles of Bowie et al. with a substitution matrix and takes into account residue pairwise interactions.

  • Elofsson A, Fischer D, Rice DW, Le Grand SM, Eisenberg D. (1996). A study of combined structure/sequence profiles.Fold Des. 1996. 1(6):451-61. [Abstract]

    For genome sequencing projects to achieve their full impact on biology and medicine, each protein sequence must be identified with its three-dimensional structure. Fold assignment methods (also called profile and threading methods) attempt to assign sequences to known protein folds by computing the compatibility of sequence to fold.

  • Bowie JU, Zhang K, Wilmanns M, Eisenberg D. (1996). Three-dimensional profiles for measuring compatibility of amino acid sequence with three-dimensional structure.Meth. Enzymol.. 1996. 266:598-616. [Abstract]
  • Bell CE, Eisenberg D. (1996). Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide.Biochemistry. Jan 1996. 35(4):1137-49. [Abstract]

    Diphtheria toxin (DT), a 58 kDa protein secreted by lysogenic strains of Corynebacterium diphtheriae, causes the disease diphtheria in humans by gaining entry into the cytoplasm of cells and inhibiting protein synthesis. Specifically, the catalytic (C) domain of DT transfers the ADP-ribose group of NAD to elongation factor-2 (EF-2), rendering EF-2 inactive. In order to investigate how the C-domain of DT binds NAD and catalyzes the ADP-ribosylation of EF-2, the crystal structure of DT in complex with NAD has been determined to 2.3 A resolution. This is the first crystal structure of an ADP-ribosyltransferase (ADP-RT) enzyme in complex with NAD and suggests the features of the ADP-RT fold which are important for NAD binding. The conformation of NAD in the complex and the proximity of the Glu148 carboxylate group of the C-domain to the scissile, N-glycosidic bond of NAD suggest plausible modes of catalysis of the ADP-ribosylation reaction. Residues 39-46 of the active-site loop of the C-domain become disordered upon NAD binding, suggesting a potential role for this loop in the recognition of the ADP-ribose acceptor substrate, EF-2. The negatively charged phosphates and two ribose hydroxyls of NAD are not in direct contact with any atoms of the C-domain. Instead, they form an exposed surface which appears to be presented for recognition by EF-2. Structural alignments of the DT-NAD complex with the structures of other members of the ADP-RT family suggest how NAD may bind to these other enzymes.

  • Fischer D, Rice D, Bowie JU, Eisenberg D. (1996). Assigning amino acid sequences to 3-dimensional protein folds.FASEB J.. Jan 1996. 10(1):126-36. [Abstract]

    With the advent of genome sequencing projects, the amino acid sequences of thousands of proteins are determined every year. Each of these protein sequences must be identified with its function and its 3-dimensional structure for us to gain a full understanding of the molecular biology of organisms. To meet this challenge, new methods are being developed for fold recognition, the computational assignment of newly determined amino acid sequences to 3-dimensional protein structures. These methods start with a library of known 3-dimensional target protein structures. The new probe sequence is then aligned to each target protein structure in the library and the compatibility of the sequence for that structure is scored. If a target structure is found to have a significantly high compatibility score, it is assumed that the probe sequence folds in much the same way as the target structure. The fundamental assumptions of this approach are that many different sequences fold in similar ways and there is a relatively high probability that a new sequence possesses a previously observed fold. We review various approaches to fold recognition and break down the process into its main steps: creation of a library of target folds; representation of the folds; alignment of the probe sequence to a target fold using a sequence-to-structure compatibility scoring function; and assessment of significance of compatibility. We emphasize that even though this new field of fold recognition has made rapid progress, technical problems remain to be solved in most of the steps. Standard benchmarks may help identify the problem steps and find solutions to the problems.

  • Ogihara NL, Parge HE, Hart PJ, Weiss MS, Goto JJ, Crane BR, Tsang J, Slater K, Roe JA, Valentine JS, Eisenberg D, Tainer JA. (1996). Unusual trigonal-planar copper configuration revealed in the atomic structure of yeast copper-zinc superoxide dismutase.Biochemistry. Feb 1996. 35(7):2316-21. [Abstract]

    The three-dimensional structure of yeast copper-zinc superoxide dismutase (CuZnSOD) has been determined in a new crystal form in space group R32 and refined against X-ray diffraction data using difference Fourier and restrained crystallographic refinement techniques. The unexpected result is that the copper ion has moved approximately 1 angstrom from its position in previously reported CuZnSOD models, the copper-imidazolate bridge is broken, and a roughly trigonal planar ligand geometry characteristic of Cu(I) rather than Cu(II) is revealed. Final R values for the two nearly identical room temperature structures are 18.6% for all 19 149 reflections in the 10.0-1.7 angstrom resolution range and 18. 2% for 17 682 reflections (F > 2 sigma) in the 10.0-1.73 angstrom resolution range. A third structure has been determined using X-ray data collected at -180 degrees C. The final R value for this structure is 19.0% (R(free) = 22.9%) for all 24 356 reflections in the 10.0-1.55 angstrom resolution range. Virtually no change in the positions of the ligands to the zinc center is observed in these models. The origin of the broken bridge and altered Cu-ligand geometry is discussed.

  • Anderson DH, Weiss MS, Eisenberg D. (1996). A challenging case for protein crystal structure determination: the mating pheromone Er-1 from Euplotes raikovi.Acta Crystallogr. D Biol. Crystallogr.. May 1996. 52(Pt 3):469-80. [Abstract]

    Four different phasing methods have been applied to the determination of the crystal structure of the 40 amino-acid mating pheromone of the unicellular ciliated protozoan Euplotes raikovi. The difficulties, failures and successes in attempts to solve the structure by: (1) molecular replacement, (2) direct phasing using the ‘Shake and Bake’ algorithm, (3) isomorphous replacement, and (4) multiple-wavelength anomalous dispersion are described. The structure was first solved by molecular replacement, and then was the first successful structure determination by ‘Shake and Bake’ without the direct involvement of its authors. A description of the current status of the high-resolution refinement of the structure is also given. The model is refined against 1 A resolution data to an R factor of 12.9%, and includes H atoms and discretely disordered side chains.

  • Fischer D, Eisenberg D. (1996). Protein fold recognition using sequence-derived predictions.Protein Sci.. May 1996. 5(5):947-55. [Abstract]

    In protein fold recognition, one assigns a probe amino acid sequence of unknown structure to one of a library of target 3D structures. Correct assignment depends on effective scoring of the probe sequence for its compatibility with each of the target structures. Here we show that, in addition to the amino acid sequence of the probe, sequence-derived properties of the probe sequence (such as the predicted secondary structure) are useful in fold assignment. The additional measure of compatibility between probe and target is the level of agreement between the predicted secondary structure of the probe and the known secondary structure of the target fold. That is, we recommend a sequence-structure compatibility function that combines previously developed compatibility functions (such as the 3D-1D scores of Bowie et al. [1991] or sequence-sequence replacement tables) with the predicted secondary structure of the probe sequence. The effect on fold assignment of adding predicted secondary structure is evaluated here by using a benchmark set of proteins (Fischer et al., 1996a). The 3D structures of the probe sequences of the benchmark are actually known, but are ignored by our method. The results show that the inclusion of the predicted secondary structure improves fold assignment by about 25%. The results also show that, if the true secondary structure of the probe were known, correct fold assignment would increase by an additional 8-32%. We conclude that incorporating sequence-derived predictions significantly improves assignment of sequences to known 3D folds. Finally, we apply the new method to assign folds to sequences in the SWISSPROT database; six fold assignments are given that are not detectable by standard sequence-sequence comparison methods; for two of these, the fold is known from X-ray crystallography and the fold assignment is correct.

  • Fahrner RL, Dieckmann T, Harwig SS, Lehrer RI, Eisenberg D, Feigon J. (1996). Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes.Chem. Biol.. Jul 1996. 3(7):543-50. [Abstract]

    The protegrins are a family of arginine- and cysteine-rich cationic peptides found in porcine leukocytes that exhibit a broad range of antimicrobial and antiviral activities. They are composed of 16-18 amino-acid residues including four cysteines, which form two disulfide linkages. To begin to understand the mechanism of action of these peptides, we set out to determine the structure of protegrin-1 (PG-1).

  • Hart PJ, Nersissian AM, Herrmann RG, Nalbandyan RM, Valentine JS, Eisenberg D. (1996). A missing link in cupredoxins: crystal structure of cucumber stellacyanin at 1.6 A resolution.Protein Sci.. Nov 1996. 5(11):2175-83. [Abstract]

    Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 A resolution. The crystallographic R-value for all 18,488 reflections (sigma > 0) between 50-1.6 A is 0.195. The overall fold is organized in two beta-sheets, both with four beta-stands. Two alpha-helices are found in loop regions between beta-strands. The beta-sheets form a beta-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the beta-barrel is more flattened, there is an extra N-terminal alpha-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.


  • Weiss MS, Blanke SR, Collier RJ, Eisenberg D. (1995). Structure of the isolated catalytic domain of diphtheria toxin.Biochemistry. Jan 1995. 34(3):773-81. [Abstract]

    The structure of the isolated catalytic domain of diphtheria toxin at pH 5.0 was determined by X-ray crystallography at 2.5 A resolution and refined to an R-factor of 19.7%. The domain is bound to its endogenous inhibitor adenylyl(3′–>5′)uridine 3′-monophosphate (ApUp). The structure of this 190-residue domain, which was expressed in and isolated from Escherichia coli, is essentially identical to the structure of the catalytic domain within whole diphtheria toxin determined at pH 7.5. However, there are two adjacent surface loops (loop 66-78 and loop 169-176) that exhibit clear differences when compared to the structure of the catalytic domain in whole diphtheria toxin. Although both loops are at the surface of the protein and are relatively flexible, the chain trace is well-defined in the electron density. The main structural difference is the closer approach of loops 66-78 and 169-176. We ascribe this structural change mainly to the absence of the neighboring transmembrane domain in the isolated catalytic domain as compared to whole diphtheria toxin. We suggest that this change represents the first step of the structural transition from the catalytic domain in whole diphtheria toxin to the translocated form of the domain. The changes are described in detail, and their implications for membrane translocation are discussed.

  • Wilmanns M, Eisenberg D. (1995). Inverse protein folding by the residue pair preference profile method: estimating the correctness of alignments of structurally compatible sequences.Protein Eng.. Jul 1995. 8(7):627-39. [Abstract]

    The residue pair preference profile (R3P) method is an inverse folding method that combines environmental profiles and pair preference profiles. The method uses statistical preferences for residue pairs which score the likelihood of finding a profiled residue to be paired with a residue within its local environment. All pairs are characterized by their dihedral angles, secondary structure and number of neighboring residues as a function of residue type. Each residue pair preference is expressed for all 20 amino acids of the profiled residue and is weighted by the compatibility of the environment residue with its own local environment. The R3P method produces an initial profile-sequence alignment which is then refined by converting the initial profile into a profile of a target sequence threaded into the structure of the initial profile. We have tested this method by evaluating alignments of sequences with known 3-D structures using structural superposition alignments as reference. R3P-sequence alignments are > or = 50% correct on average for sequences whose 3-D structure pairs superimpose with an r.m.s. deviation of < or = 1.97 A. The average improvement in correctness during this iterative refinement is 14%. The R3P-sequence alignments are compared with sequence-sequence and 3-D profile-sequence alignments. When all three methods are combined, on average > or = 50% of the alignments are correct for pairs of 3-D structures that superimpose within 2.12 A. A 3-D model of HisA is predicted with the combined method.

  • Vrielink A, Beamer L, Le T, Eisenberg D. (1995). Crystallization of the chaperone protein SecB.Protein Sci.. Aug 1995. 4(8):1651-3. [Abstract]

    The secretory protein SecB found in Escherichia coli is a molecular chaperone that binds to precursor forms of a number of proteins targeted for export to the periplasmic space. SecB maintains these proteins in a translocation-competent conformation facilitating the translocation process. The material has been cloned and expressed in E. coli. Crystals have been grown from polyethylene glycol 8000 by vapor diffusion using the hanging drop technique. These crystals are monoclinic, belonging to space group C2 with unit cell dimensions a = 56.0 A, b = 111.1 A, c = 134.7 A, and beta = 104 degrees. The crystals diffract to 8 A resolution on a Rigaku imaging plate detector. Dynamic light scattering experiments suggest that SecB exhibits aggregation behavior with a number of different precipitating agents. These results may explain resistance of SecB to forming ordered crystals.

  • Elofsson A, Le Grand SM, Eisenberg D. (1995). Local moves: an efficient algorithm for simulation of protein folding.Proteins. Sep 1995. 23(1):73-82. [Abstract]

    We have enhanced genetic algorithms and Monte Carlo methods for simulation of protein folding by introducing “local moves” in dihedral space. A local move consists of changes in backbone dihedral angles in a sequential window while the positions of all atoms outside the window remain unchanged. We find three advantages of local moves: (1) For some energy functions, protein conformations of lower energy are found; (2) these low energy conformations are found in fewer steps; and (3) the simulations are less sensitive to the details of the annealing protocol. To distinguish the effectiveness of local move algorithm from the complexity of the energy function, we have used several different energy functions. These energy functions include the Profile score (Bowie et al., Science 253:164-170, 1991), the knowledge-based energy function used by Bowie and Eisenberg 1994 (Proc. Natl. Acad. Sci. U.S.A. 91:4434-4440, 1994), two energy terms developed as suggested by Sippl and coworkers (Hendlich et al., J. Mol. Biol. 216:167-180, 1990), and AMBER (Weiner and Kollman, J. Comp. Chem. 2:287-303, 1981). Besides these energy functions we have used three energy functions that include knowledge of the native structures: the RMSD from the native structure, the distance matrix error, and an energy term based on the distance between different residue types called DBIN. In some of these simulations the main advantage of local moves is the reduced dependence on the details of the annealing schedule. In other simulations, local moves are superior to other algorithms as structures with lower energy are found.

  • Weiss MS, Anderson DH, Raffioni S, Bradshaw RA, Ortenzi C, Luporini P, Eisenberg D. (1995). A cooperative model for receptor recognition and cell adhesion: evidence from the molecular packing in the 1.6-A crystal structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.Proc. Natl. Acad. Sci. U.S.A.. Oct 1995. 92(22):10172-6. [Abstract]

    The crystal structure of the pheromone Er-1 from the unicellular eukaryotic organism Euplotes raikovi was determined at 1.6 A resolution and refined to a crystallographic R factor of 19.9%. In the tightly packed crystal, two extensive intermolecular helix-helix interactions arrange the Er-1 molecules into layers. Since the putative receptor of the pheromone is a membrane-bound protein, whose extracellular C-terminal domain is identical in amino acid sequence to the soluble pheromone, the interactions found in the crystal may mimic the pheromone-receptor interactions as they occur on a cell surface. Based on this, we propose a model for the interaction between soluble pheromone molecules and their receptors. In this model, strong pheromone-receptor binding emerges as a consequence of the cooperative utilization of several weak interactions. The model offers an explanation for the results of binding studies and may also explain the adhesion between cells that occurs during mating.

  • Bennett MJ, Schlunegger MP, Eisenberg D. (1995). 3D domain swapping: a mechanism for oligomer assembly.Protein Sci.. Dec 1995. 4(12):2455-68. [Abstract]

    3D domain swapping is a mechanism for forming oligomeric proteins from their monomers. In 3D domain swapping, one domain of a monomeric protein is replaced by the same domain from an identical protein chain. The result is an intertwined dimer or higher oligomer, with one domain of each subunit replaced by the identical domain from another subunit. The swapped “domain” can be as large as an entire tertiary globular domain, or as small as an alpha-helix or a strand of a beta-sheet. Examples of 3D domain swapping are reviewed that suggest domain swapping can serve as a mechanism for functional interconversion between monomers and oligomers, and that domain swapping may serve as a mechanism for evolution of some oligomeric proteins. Domain-swapped proteins present examples of a single protein chain folding into two distinct structures.


  • Liaw SH, Eisenberg D. (1994). Structural model for the reaction mechanism of glutamine synthetase, based on five crystal structures of enzyme-substrate complexes.Biochemistry. Jan 1994. 33(3):675-81. [Abstract]

    Glutamine synthetase brings nitrogen into metabolism by condensing ammonia and glutamate, with the aid of ATP, to yield glutamine, ADP, and inorganic phosphate. Here we present five crystal structures of GS complexed with each of two substrates, Glu and AMPPNP (an ATP analog), with a transition-state analogue, L-methionine-S-sulfoximine, and with each of two products, Gln and ADP. GS of the present study is from Salmonella typhimurium, has Mn2+ bound, and is fully unadenylylated. Protein-metal-substrate interactions and small but significant conformational changes induced by substrate binding are defined by Fourier maps. On the basis of these maps, we propose a tentative structure-based enzymatic mechanism of glutamine synthesis with these steps: (1) ATP binds first at the top of the funnel-shaped active site cavity, adjacent to the n2 Mn2+; Arg 359 moves toward the Glu binding site. (2) Glu binds adjacent to the n1 Mn2+ at the bottom of the active site near a flexible loop (residues 324-328). As proposed earlier by Meister and others, Glu attacks the gamma-phosphorus atom of ATP to produce gamma-glutamyl phosphate and ADP. (3) The presence of ADP (but not ATP) moves Arg 339 toward the Pi site, perhaps stabilizing the gamma-glutamyl phosphate, and moves Asp 50′ of the adjacent subunit toward a putative ammonium ion site, enhancing binding of this third substrate. Deprotonation of the ammonium ion, perhaps by Asp 50′, permits the resulting active species, ammonia, to attack the gamma-glutamyl phosphate, forming a tetrahedral intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

  • Zhang KY, Cascio D, Eisenberg D. (1994). Crystal structure of the unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate.Protein Sci.. Jan 1994. 3(1):64-9. [Abstract]

    The crystal structure of unactivated ribulose 1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum complexed with a transition state analog, 2-carboxy-D-arabinitol 1,5-bisphosphate, was determined to 2.7 A resolution by X-ray crystallography. The transition state analog binds at the active site in an extended conformation. As compared to the binding of the same analog in the activated enzyme, the analog binds in a reverse orientation. The active site Lys 201 is within hydrogen bonding distance of the carboxyl oxygen of the analog. Loop 6 (residues 330-339) remains open and flexible upon binding of the analog in the unactivated enzyme, in contrast to the closed and ordered loop 6 in the activated enzyme complex. The transition state analog is exposed to solvent due to the open conformation of loop 6.

  • Bennett MJ, Choe S, Eisenberg D. (1994). Domain swapping: entangling alliances between proteins.Proc. Natl. Acad. Sci. U.S.A.. Apr 1994. 91(8):3127-31. [Abstract]

    The comparison of monomeric and dimeric diphtheria toxin (DT) reveals a mode for protein association which we call domain swapping. The structure of dimeric DT has been extensively refined against data to 2.0-A resolution and a three-residue loop has been corrected as compared with our published 2.5-A-resolution structure. The monomeric DT structure has also been determined, at 2.3-A resolution. Monomeric DT is a Y-shaped molecule with three domains: catalytic (C), transmembrane (T), and receptor binding (R). Upon freezing in phosphate buffer, DT forms a long-lived, metastable dimer. The protein chain tracing discloses that upon dimerization an unprecedented conformational rearrangement occurs: the entire R domain from each molecule of the dimer is exchanged for the R domain from the other. This involves breaking the noncovalent interactions between the R domain and the C and T domains, rotating the R domain by 180 degrees with atomic movements up to 65 A, and re-forming the same noncovalent interactions between the R domain and the C and T domains of the other chain of the dimer. This conformational transition explains the long life and metastability of the DT dimer. Several other intertwined, dimeric protein structures satisfy our definition of domain swapping and suggest that domain swapping may be the molecular mechanism for evolution of these oligomers and possibly of oligomeric proteins in general.

  • McKenna MC, Muchardt C, Gaynor R, Eisenberg D. (1994). Preparative scale culture of Escherichia coli cells expressing the human immunodeficiency virus type 1 Tat protein.Protein Expr. Purif.. Apr 1994. 5(2):105-11. [Abstract]

    A procedure leading to a 100-liter fermentor culture of Escherichia coli cells expressing the human immunodeficiency virus type 1 (HIV-1) trans-activator (Tat) protein is described. The effects of growth temperature and of cell density at the time of induction on the yield of Tat were investigated. Tat was identified by SDS-gel electrophoresis and Western blot. Tat represents approximately 10% of the soluble protein in the cell lysate.

  • Zhang KY, Eisenberg D. (1994). The three-dimensional profile method using residue preference as a continuous function of residue environment.Protein Sci.. Apr 1994. 3(4):687-95. [Abstract]

    In the 3-dimensional profile method, the compatibility of an amino acid sequence for a given protein structure is scored as the sum of the preferences of the residues for their environments in the 3D structure. In the original method (Bowie JU, Lüthy R, Eisenberg D, 1991, Science 253:164-170), residue environments were quantized into 18 discrete environmental classes. Here, amino acid residue preferences are expressed as a continuous function of environmental variables (residue area buried and fractional area buried by polar atoms). This continuous representation of residue preferences, expressed as a Fourier series, avoids the abrupt change of preference of residues in slightly different environments, as encountered in the original method with its 18 discrete environmental classes. When compared with the discrete 18-class representation of residue environments, this continuous 3D profile is found to be more sensitive in identifying sequences that fold into the profiled structure but share with it little sequence identity. The continuous 3D profile is also less sensitive to errors in environmental variables than is the discrete 3D profile. The continuous 3D profile can also be used to detect wrong folds or incorrectly modeled segments in an otherwise correct structure, as could the discrete 3D profile (Lüthy R, Bowie JU, Eisenberg D, 1992, Nature 356:83-85). Moreover, the progress of structure improvement during atomic refinement can also be monitored by examining the profile scores in a moving-window scan. Finally, by defining a functional form for profile scores, we open the way to profile atomic refinement in which an atomic structure adjusts to produce residue environments more compatible with the protein side chains.

  • Bowie JU, Eisenberg D. (1994). An evolutionary approach to folding small alpha-helical proteins that uses sequence information and an empirical guiding fitness function.Proc. Natl. Acad. Sci. U.S.A.. May 1994. 91(10):4436-40. [Abstract]

    Three short protein sequences have been guided by computer to folds resembling their crystal structures. Initially, peptide fragment conformations ranging in size from 9 to 25 residues were selected from a database of known protein structures. A fragment was selected if it was compatible with a segment of the sequence to be folded, as judged by three-dimensional profile scores. By linking the selected fragment conformations together, hundreds of trial structures were generated of the same length and sequence as the protein to be folded. These starting trial structures were then improved by an evolutionary algorithm. Selection pressure for improving the structures was provided by an energy function that was designed to guide the conformational search procedure toward the correct structure. We find that by evolution of only 400 structures for fewer than 1400 generations, the overall fold of some small helical proteins can be computed from the sequence, with deviations from observed structures of 2.5-4.0 A for C alpha atoms.

  • Zhang KY, Eisenberg D. (1994). Solid-state phase transition in the crystal structure of ribulose 1,5-bisphosphate carboxylase/oxygenase.Acta Crystallogr. D Biol. Crystallogr.. May 1994. 50(Pt 3):258-62. [Abstract]

    The crystal structure is described of ribulose 1,5-bisphosphate carboxylase/oxygenase in a new crystal form. This new form (form V) was obtained from a previously known crystal form (form III) through a solid-state phase transition. The solid-state phase transition was brought about by transferring the crystal from a high-salt low-pH mother liquor to a low-salt high-pH synthetic mother liquor. The interplay of electrostatic repulsion and osmotic pressure induced a unit-cell shrinkage of 24 A along the c axis and expansion of 4 A along the a and b axes. The space group also changed from I422 to I4. The new crystal form was shown to be more resistant to X-ray radiation damage, which suggests the effect of crystal stabilization by non-penetrating molecules. The structure of ribulose 1,5-bisphosphate carboxylase/oxygenase in the new crystal form is compared with that of the old crystal form.

  • McKenna MC, Anderson D, Cascio D, Eisenberg D. (1994). Crystallization studies of the human immunodeficiency virus (HIV-1) Tat protein and its trans-activation response element (TAR) RNA.Acta Crystallogr. D Biol. Crystallogr.. Jul 1994. 50(Pt 4):527-34. [Abstract]

    Small single crystals are reported of a complex between a small peptide fragment of the HIV-1 Tat protein and a fragment of the RNA to which it binds. Tat is responsible for enhancing the level of expression of the human immunodeficiency virus type 1 (HIV-1) and is a logical target for AIDS therapy. Tat may function to increase the level of transcription initiation or to prevent premature termination of transcripts. In vitro, Tat binds through its basic domain (two Lys and six Arg in nine residues) to a three-nucleotide bulge of a stem-loop RNA structure called TAR. Complex formation between Tat and TAR is necessary for Tat activity. Peptides which contain the basic region of Tat also bind to TAR RNA. We have carried out crystallization experiments on a 27-nucleotide fragment of TAR RNA and on complexes between two Tat peptides and TAR.

  • Privé GG, Verner GE, Weitzman C, Zen KH, Eisenberg D, Kaback HR. (1994). Fusion proteins as tools for crystallization: the lactose permease from Escherichia coli.Acta Crystallogr. D Biol. Crystallogr.. Jul 1994. 50(Pt 4):375-9. [Abstract]

    A novel strategy is presented for the crystallization of membrane proteins or other proteins with low solubility and/or stability. The method is illustrated with the lactose permease from Escherichia coli, in which a fusion is constructed between the permease and a ‘carrier’ protein. The carrier is a soluble, stable protein with its C and N termini close together in space at the surface of the protein, so that the carrier can be introduced into an internal position of the target protein. The carrier is chosen with convenient spectral or enzymatic properties, making the fusion protein easier to handle than the native molecule. Data are presented for the successful construction, expression and purification of a fusion product between lactose permease and cytochrome b(562) from E. coli. The lactose transport activity of the fusion protein is similar to that of wild-type lactose permease, and the fusion product has an absorption spectrum in the visible range which is essentially identical to that of cytochrome b(562). The fusion protein has a higher proportional polar surface area than wild-type permease, and should have better possibilities of forming the strong directional intermolecular contacts required of a crystal lattice.

  • Bennett MJ, Eisenberg D. (1994). Refined structure of monomeric diphtheria toxin at 2.3 A resolution.Protein Sci.. Sep 1994. 3(9):1464-75. [Abstract]

    The structure of toxic monomeric diphtheria toxin (DT) was determined at 2.3 A resolution by molecular replacement based on the domain structures in dimeric DT and refined to an R factor of 20.7%. The model consists of 2 monomers in the asymmetric unit (1,046 amino acid residues), including 2 bound adenylyl 3′-5′ uridine 3′ monophosphate molecules and 396 water molecules. The structures of the 3 domains are virtually identical in monomeric and dimeric DT; however, monomeric DT is compact and globular as compared to the “open” monomer within dimeric DT (Bennett MJ, Choe S, Eisenberg D, 1994b, Protein Sci 3:0000-0000). Detailed differences between monomeric and dimeric DT are described, particularly (1) changes in main-chain conformations of 8 residues acting as a hinge to “open” or “close” the receptor-binding (R) domain, and (2) a possible receptor-docking site, a beta-hairpin loop protruding from the R domain containing residues that bind the cell-surface DT receptor. Based on the monomeric and dimeric DT crystal structures we have determined and the solution studies of others, we present a 5-step structure-based mechanism of intoxication: (1) proteolysis of a disulfide-linked surface loop (residues 186-201) between the catalytic (C) and transmembrane (T) domains; (2) binding of a beta-hairpin loop protruding from the R domain to the DT receptor, leading to receptor-mediated endocytosis; (3) low pH-triggered open monomer formation and exposure of apolar surfaces in the T domain, which insert into the endosomal membrane; (4) translocation of the C domain into the cytosol; and (5) catalysis by the C domain of ADP-ribosylation of elongation factor 2.

  • Bennett MJ, Choe S, Eisenberg D. (1994). Refined structure of dimeric diphtheria toxin at 2.0 A resolution.Protein Sci.. Sep 1994. 3(9):1444-63. [Abstract]

    The refined structure of dimeric diphtheria toxin (DT) at 2.0 A resolution, based on 37,727 unique reflections (F > 1 sigma (F)), yields a final R factor of 19.5% with a model obeying standard geometry. The refined model consists of 523 amino acid residues, 1 molecule of the bound dinucleotide inhibitor adenylyl 3′-5′ uridine 3′ monophosphate (ApUp), and 405 well-ordered water molecules. The 2.0-A refined model reveals that the binding motif for ApUp includes residues in the catalytic and receptor-binding domains and is different from the Rossmann dinucleotide-binding fold. ApUp is bound in part by a long loop (residues 34-52) that crosses the active site. Several residues in the active site were previously identified as NAD-binding residues. Glu 148, previously identified as playing a catalytic role in ADP-ribosylation of elongation factor 2 by DT, is about 5 A from uracil in ApUp. The trigger for insertion of the transmembrane domain of DT into the endosomal membrane at low pH may involve 3 intradomain and 4 interdomain salt bridges that will be weakened at low pH by protonation of their acidic residues. The refined model also reveals that each molecule in dimeric DT has an “open” structure unlike most globular proteins, which we call an open monomer. Two open monomers interact by “domain swapping” to form a compact, globular dimeric DT structure. The possibility that the open monomer resembles a membrane insertion intermediate is discussed.

  • Eisenberg D. (1994). Max Perutz’s achievements: how did he do it?Protein Sci.. Oct 1994. 3(10):1625-8. [Abstract]


  • Lovejoy B, Choe S, Cascio D, McRorie DK, DeGrado WF, Eisenberg D. (1993). Crystal structure of a synthetic triple-stranded alpha-helical bundle.Science. Feb 1993. 259(5099):1288-93. [Abstract]

    The x-ray crystal structure of a peptide designed to form a double-stranded parallel coiled coil shows that it is actually a triple-stranded coiled coil formed by three alpha-helices. Unlike the designed parallel coiled coil, the helices run up-up-down. The structure is stabilized by a distinctive hydrophobic interface consisting of eight layers. As in the design, each alpha-helix in the coiled coil contributes one leucine side chain to each layer. The structure suggests that hydrophobic interactions are a dominant factor in the stabilization of coiled coils. The stoichiometry and geometry of coiled coils are primarily determined by side chain packing in the solvent-inaccessible interior, but electrostatic interactions also contribute.

  • Wilmanns M, Eisenberg D. (1993). Three-dimensional profiles from residue-pair preferences: identification of sequences with beta/alpha-barrel fold.Proc. Natl. Acad. Sci. U.S.A.. Feb 1993. 90(4):1379-83. [Abstract]

    The three-dimensional profile method expresses the three-dimensional structure of a protein as a table, the profile, which represents the local environment of each residue. The score of an amino acid sequence, aligned with the three-dimensional profile, reflects its compatibility with the profiled structure. In the original implementation, each local environment was characterized by its polarity, the area buried of its side chain, and its secondary structure. Here we describe a modified three-dimensional profile algorithm that characterizes the local environment in terms of the statistical preferences of the profiled residue for neighbors of specific residue types, main-chain conformations, or secondary structure. Combined profiles of the original and the three new types were tested on beta/alpha-barrel protein structures. The method identified the following enzymes of unknown three-dimensional structure as probable beta/alpha-barrels, all of which catalyze reactions in the biosynthesis of aromatic amino acids: anthranilate phosphoribosyltransferase (trpD), glutamine amidotransferase (trpG), and phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA).

  • Liaw SH, Jun G, Eisenberg D. (1993). Extending the diffraction limit of protein crystals: the example of glutamine synthetase from Salmonella typhimurium in the presence of its cofactor ATP.Protein Sci.. Mar 1993. 2(3):470-1. [Abstract]
  • Liaw SH, Pan C, Eisenberg D. (1993). Feedback inhibition of fully unadenylylated glutamine synthetase from Salmonella typhimurium by glycine, alanine, and serine.Proc. Natl. Acad. Sci. U.S.A.. Jun 1993. 90(11):4996-5000. [Abstract]

    Bacterial glutamine synthetase (GS; EC was previously shown to be inhibited by nine end products of glutamine metabolism. Here we present four crystal structures of GS, complexed with the substrate Glu and with each of three feedback inhibitors. The GS of the present study is from Salmonella typhimurium, with Mn2+ ions bound, and is fully unadenylylated. From Fourier difference maps, we find that L-serine, L-alanine, and glycine bind at the site of the substrate L-glutamate. In our model, these four amino acids bind with the atoms they share in common (the “main chain” +NH3-CH-COO-) in the same positions. Thus on the basis of our x-ray work, glycine, alanine, and serine appear to inhibit GS-Mn by competing with the substrate glutamate for the active site.

  • Hill CP, Osslund TD, Eisenberg D. (1993). The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors.Proc. Natl. Acad. Sci. U.S.A.. Jun 1993. 90(11):5167-71. [Abstract]

    We have determined the three-dimensional structure of recombinant human granulocyte-colony-stimulating factor by x-ray crystallography. Phases were initially obtained at 3.0-A resolution by multiple isomorphous replacement and were refined by solvent flattening and by averaging of the electron density of the three molecules in the asymmetric unit. The current R factor is 21.5% for all data between 6.0- and 2.2-A resolution. The structure is predominantly helical, with 104 of the 175 residues forming a four-alpha-helix bundle. The only other secondary structure is also helical. In the loop between the first two long helices a four-residue 3(10)-helix is immediately followed by a 6-residue alpha-helix. Three residues in the short connection between the second and third bundle helices form almost one turn of left-handed helix. The up-up-down-down connectivity with two long crossover connections has been reported previously for five other proteins, which like granulocyte-colony-stimulating factor are all signaling ligands: growth hormone, granulocyte/macrophage-colony-stimulating factor, interferon beta, interleukin 2, and interleukin 4. Structural similarity among these growth factors occurs despite the absence of similarity in their amino acid sequences. Conservation of this tertiary structure suggests that these different growth factors might all bind to their respective sequence-related receptors in an equivalent manner.

  • Schreuder HA, Knight S, Curmi PM, Andersson I, Cascio D, Sweet RM, Brändén CI, Eisenberg D. (1993). Crystal structure of activated tobacco rubisco complexed with the reaction-intermediate analogue 2-carboxy-arabinitol 1,5-bisphosphate.Protein Sci.. Jul 1993. 2(7):1136-46. [Abstract]

    The crystal structure of activated tobacco rubisco, complexed with the reaction-intermediate analogue 2-carboxy-arabinitol 1,5-bisphosphate (CABP) has been determined by molecular replacement, using the structure of activated spinach rubisco (Knight, S., Andersson, I., & Brändén, C.-I., 1990, J. Mol. Biol. 215, 113-160) as a model. The R-factor after refinement is 21.0% for 57,855 reflections between 9.0 and 2.7 A resolution. The local fourfold axis of the rubisco hexadecamer coincides with a crystallographic twofold axis. The result is that the asymmetric unit of the crystals contains half of the L8S8 complex (molecular mass 280 kDa in the asymmetric unit). The activated form of tobacco rubisco is very similar to the activated form of spinach rubisco. The root mean square difference is 0.4 A for 587 equivalent C alpha atoms. Analysis of mutations between tobacco and spinach rubisco revealed that the vast majority of mutations concerned exposed residues. Only 7 buried residues were found to be mutated versus 54 residues at or near the surface of the protein. The crystal structure suggests that the Cys 247-Cys 247 and Cys 449-Cys 459 pairs are linked via disulfide bridges. This pattern of disulfide links differ from the pattern of disulfide links observed in crystals of unactivated tobacco rubisco (Curmi, P.M.G., et al., 1992, J. Biol. Chem. 267, 16980-16989) and is similar to the pattern observed for activated spinach tobacco.

  • Liaw SH, Villafranca JJ, Eisenberg D. (1993). A model for oxidative modification of glutamine synthetase, based on crystal structures of mutant H269N and the oxidized enzyme.Biochemistry. Aug 1993. 32(31):7999-8003. [Abstract]

    Proteolytic degradation of glutamine synthetase (GS) in Escherichia coli is known to follow “marking” by oxidative modification. At an early stage of the degradative pathway, oxidation of His 269 and Arg 344 abolishes GS enzymatic activity. We propose a mechanism for the early stage of oxidative inactivation of GS on the basis of the crystal structure of H269N and tryptophan fluorescence spectra of H269N and H269NR344Q: (1) Oxidation of Arg 344, adjacent to the n2 metal ion site, decreases ATP binding. (2) Oxidation of His 269 to Asn destroys the n2 site, consistent with the function of His 269 as a ligand for the n2 metal. (3) Loss of Mn2+ at the n2 site destroys the integrity of the ATP binding site. (4) Destruction of the ATP site results in the observed low enzymatic activity of H269N and H269NR344Q. During later stages of oxidative modification, the n1 metal ion site is destroyed and the active site of the enzyme becomes flexible as suggested by X-ray data collected from an oxidized crystal of GS. Thus, studies of mutant and oxidized enzymes confirm that there are at least two stages of oxidative modification of GS. These studies suggest that the early modification occurs at the n2 metal ion site, eliminating enzyme activity, and the later modification occurs at the n1 metal ion site, relaxing the GS structure, perhaps enabling proteolytic degradation. These studies also illuminate the differing roles of the two bound metal ions: the tightly bound n1 ion enhances the stability of the catalytically active conformation, and the less tightly bound n2 ion participates in ATP binding.

  • Fujii G, Selsted ME, Eisenberg D. (1993). Defensins promote fusion and lysis of negatively charged membranes.Protein Sci.. Aug 1993. 2(8):1301-12. [Abstract]

    Defensins, a family of cationic peptides isolated from mammalian granulocytes and believed to permeabilize membranes, were tested for their ability to cause fusion and lysis of liposomes. Unlike alpha-helical peptides whose lytic effects have been extensively studied, the defensins consist primarily of beta-sheet. Defensins fuse and lyse negatively charged liposomes but display reduced activity with neutral liposomes. These and other experiments suggest that fusion and lysis is mediated primarily by electrostatic forces and to a lesser extent, by hydrophobic interactions. Circular dichroism and fluorescence spectroscopy of native defensins indicate that the amphiphilic beta-sheet structure is maintained throughout the fusion process. Taken together, these results support the idea that protein-mediated membrane fusion depends not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form a three-dimensional amphiphilic structure, which promotes the efficient mixing of the lipids between membranes. A molecular model for membrane fusion by defensins is presented, which takes into account the contributions of electrostatic forces, hydrophobic interactions, and structural amphiphilicity.

  • Schreuder HA, Knight S, Curmi PM, Andersson I, Cascio D, Brändén CI, Eisenberg D. (1993). Formation of the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase by a disorder-order transition from the unactivated to the activated form.Proc. Natl. Acad. Sci. U.S.A.. Nov 1993. 90(21):9968-72. [Abstract]

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the key first step in photosynthetic CO2 fixation, the reaction that incorporates CO2 into sugar. In this study, refined crystal structures of unactivated tobacco RuBisCO and activated RuBisCO from spinach and tobacco, in complex with the reaction-intermediate analog 2-carboxyarabinitol 1,5-bisphosphate (CABP), are compared. Both plant enzymes are hexadecameric complexes of eight large and eight small subunits with a total relative molecular mass of approximately 550,000. The comparison of activated and unactivated forms of RuBisCO provides insight into the dynamics of action of this enzyme. The catalytic site, which is open to the solvent in the unactivated enzyme, becomes shielded in the activated CABP complex. This shielding is accomplished by a 12-A movement of the active-site “loop 6” (residues 331-338) and a disorder-order transition of three loops near the active-site entrance, the N terminus, the C terminus, and a loop comprising residues 64-68. All these residues belong to the catalytic large subunit. Domain rotations of about 2 degrees are observed, also tightening the active-site cleft. These observations provide an explanation for the extremely tight binding (Kd < or = 10(-11) M) of the CABP molecule. A striking correlation exists between crystallographic temperature factors in the activated enzyme and the magnitude of the atomic movement upon activation.

  • Lovejoy B, Cascio D, Eisenberg D. (1993). Crystal structure of canine and bovine granulocyte-colony stimulating factor (G-CSF).J. Mol. Biol.. Dec 1993. 234(3):640-53. [Abstract]

    The crystal structures of recombinant canine and bovine granulocyte colony stimulating factor (G-CSF) have been determined by X-ray crystallography, using molecular replacement with recombinant human G-CSF as a model. G-CSF is a member of the cytokine family of glycoproteins that stimulate the differentiation and proliferation of blood cells. Human, bovine and canine G-CSF all have a molecular mass of about 19 kDa and share an amino acid sequence identity of about 80%. Two crystal forms of canine G-CSF have been solved. Form I recombinant canine G-CSF (rcG-CSFI; space group C2) contains one molecule in the asymmetric unit while form II canine G-CSF (rcG-CSFII; space group P2(1)) has two molecules in the asymmetric unit and bovine G-CSF (rbG-CSF; space group P2(1)2(1)2(1)) contains one molecule in the asymmetric unit. rcG-CSFI has been refined to an R factor of 20.7% with data to 2.3 A resolution and rcG-CSFII has been refined to an R factor of 19.3% with data to 2.2 A resolution. rbG-CSF has been refined to an R factor of 21.3% with data to 1.7 A resolution. The structure of human, canine and bovine G-CSF is an antiparallel 4-alpha-helical bundle with up-up-down-down connectivity. With the exception of one highly exposed loop (residues 66 to 74), the human, canine and bovine structures are very similar to each other. Using our series of G-CSF crystal structures we developed a function that describes the probability that a particular residue position (i) contributes to a G-CSF receptor binding site based on two principles, (1) high sequence conservation in the primary sequence of human, bovine, canine and murine G-CSF and (2) conservation of high solvent accessibility in the human, bovine and canine crystal structures. On the basis of this probability function as well as a comparison of G-CSF to the crystal structure of human growth hormone (hGH) complexed with the extracellular domain of the human growth hormone receptor (hGHbp), residues that contribute to potential G-CSF receptor binding sites are identified.


  • Eisenberg D, Bowie JU, Lüthy R, Choe S. (1992). Three-dimensional profiles for analysing protein sequence-structure relationships.Faraday Discuss.. 1992. (93):25-34. [Abstract]

    In the method of 3D (three-dimensional) profiles, each residue position in a protein is characterized by its environment and is represented by a row of 20 numbers in a table, the profile. These numbers are the statistical preferences (called 3D-1D scores) of each of the 20 amino acids for this environment. A profile is computed from the coordinates of a protein model, and it gives a score S for any amino acid sequence folded as the model. To date 3D profiles have found three applications. The first is to identify other protein sequences which are folded in the same general pattern as the structure from which the profile was prepared. These are sequences which have high scores for the profile computed from the model. The second is to assess the validity of protein models, however determined. Correct models are found to give profiles that have high scores for their own amino acid sequences, and incorrect models are found to have lower scores. The example of the X-ray structure determination of diphtheria toxin is discussed. The third application is to assess which is the stable oligomeric state of a folded protein. Several examples suggest that the highest profile score for a sequence is achieved when the protein is aggregated into its most stable oligomeric state.

  • Wesson L, Eisenberg D. (1992). Atomic solvation parameters applied to molecular dynamics of proteins in solution.Protein Sci.. Feb 1992. 1(2):227-35. [Abstract]

    A solvation energy function for use in the molecular simulation of proteins is proposed. It is based on the accessible surface areas of atoms in the protein and on atomic solvation parameters derived from empirical vapor-to-water free energies of transfer of amino acid side-chain analogs. The energy function and its derivatives were added to the CHARMM molecular simulation program (Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., & Karplus, M., 1983, J. Comput. Chem. 4(2), 187-217). The effect of the added energy term was evaluated by 110 ps of molecular dynamics on the 26-residue protein melittin. The melittin monomer and tetramer were studied both with and without the added term. With the added energy term the monomer partially unfolded, while the secondary structure of the tetramer was preserved, in agreement with reported experiments (Brown, L.R., Lauterwein, J., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 231-244; Lauterwein, J., Brown, L.R., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 219-230).

  • Lüthy R, Bowie JU, Eisenberg D. (1992). Assessment of protein models with three-dimensional profiles.Nature. Mar 1992. 356(6364):83-5. [Abstract]

    As methods for determining protein three-dimensional (3D) structure develop, a continuing problem is how to verify that the final protein model is correct. The revision of several protein models to correct errors has prompted the development of new criteria for judging the validity of X-ray and NMR structures, as well as the formation of energetic and empirical methods to evaluate the correctness of protein models. The challenge is to distinguish between a mistraced or wrongly folded model, and one that is basically correct, but not adequately refined. We show that an effective test of the accuracy of a 3D protein model is a comparison of the model to its own amino-acid sequence, using a 3D profile, computed from the atomic coordinates of the structure 3D profiles of correct protein structures match their own sequences with high scores. In contrast, 3D profiles for protein models known to be wrong score poorly. An incorrectly modelled segment in an otherwise correct structure can be identified by examining the profile score in a moving-window scan. The accuracy of a protein model can be assessed by its 3D profile, regardless of whether the model has been derived by X-ray, NMR or computational procedures.

  • Choe S, Bennett MJ, Fujii G, Curmi PM, Kantardjieff KA, Collier RJ, Eisenberg D. (1992). The crystal structure of diphtheria toxin.Nature. May 1992. 357(6375):216-22. [Abstract]

    The crystal structure of the diphtheria toxin dimer at 2.5 A resolution reveals a Y-shaped molecule of three domains. The catalytic domain, called fragment A, is of the alpha + beta type. Fragment B actually consists of two domains. The transmembrane domain consists of nine alpha-helices, two pairs of which are unusually apolar and may participate in pH-triggered membrane insertion and translocation. The receptor-binding domain is a flattened beta-barrel with a jelly-roll-like topology. Three distinct functions of the toxin, each carried out by a separate structural domain, can be useful in designing chimaeric proteins, such as immunotoxins, in which the receptor-binding domain is substituted with antibodies to target other cell types.

  • Wilcox W, Eisenberg D. (1992). Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding.Protein Sci.. May 1992. 1(5):641-53. [Abstract]

    The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed “folding”) are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.

  • Lovejoy B, Le TC, Lüthy R, Cascio D, O’Neil KT, DeGrado WF, Eisenberg D. (1992). X-ray grade crystals of a designed alpha-helical coiled coil.Protein Sci.. Jul 1992. 1(7):956-7. [Abstract]
  • Curmi PM, Cascio D, Sweet RM, Eisenberg D, Schreuder H. (1992). Crystal structure of the unactivated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from tobacco refined at 2.0-A resolution.J. Biol. Chem.. Aug 1992. 267(24):16980-9. [Abstract]

    The structure of the unactivated form of ribulose-1,5-bisphosphate carboxylase/oxygenase was refined at a resolution of 2.0 A to an R-factor of 17.1%. The previous model (Chapman et al., 1988) was extensively rebuilt, and the small subunit was retraced. The refined model consists of residues 22-63 and 69-467 of the large subunit and the complete small subunit. A striking feature of the model is that several loops have very high B-factors, probably representing mobile regions of the molecule. An examination of the intersubunit contacts shows that the L8S8 hexadecamer is composed of four L2 dimers. The dominant contacts between these L2 dimers are formed by the small subunits. This suggests that the small subunits may be essential for maintaining the integrity of the L8S8 structure. The active site shows differences between the unactivated form and the quaternary complex. In particular, Lys334 has moved out of the active site by about 10A. This residue lies on loop 6 of the alpha beta barrel, which is a particularly mobile loop. The site of ribulose-1,5-bisphosphate carboxylase/oxygenase activation is well ordered in the absence of the carbamylation of Lys201 and Mg2+ binding. The residues are held poised by a network of hydrogen bonds. In the unactivated state, the active site is accessible to substrate binding.

  • Liu Y, Peter D, Roghani A, Schuldiner S, Privé GG, Eisenberg D, Brecha N, Edwards RH. (1992). A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter.Cell. Aug 1992. 70(4):539-51. [Abstract]

    Classical neurotransmitters are transported into synaptic vesicles so that their release can be regulated by neural activity. In addition, the vesicular transport of biogenic amines modulates susceptibility to N-methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin N-methyl-1,2,3,6-tetrahydropyridine that produces a model of Parkinson’s disease. Taking advantage of selection in MPP+, we have used gene transfer followed by plasmid rescue to identify a cDNA clone that encodes a vesicular amine transporter. The sequence predicts a novel mammalian protein with 12 transmembrane domains and homology to a class of bacterial drug resistance transporters. We have detected messenger RNA transcripts for this transporter only in the adrenal gland. Monoamine cell populations in the brain stem express a distinct but highly related protein.

  • Lovejoy B, Akerfeldt KS, DeGrado WF, Eisenberg D. (1992). Crystallization of proton channel peptides.Protein Sci.. Aug 1992. 1(8):1073-7. [Abstract]

    Crystals have been grown of two similar peptides that form ion-conducting channels in diphytanoyl phosphatidylcholine bilayers. These crystals were grown by slow evaporation of the organic solvent, 2,2,2-trifluoroethanol. Crystals of one of the peptides have been characterized by X-ray diffraction, and X-ray data have been measured to 2.3 A resolution. Earlier it was proposed that the ion-conducting channels formed by these peptides consist of four peptides associated as a parallel alpha-helical tetramer. On the basis of the space group and unit cell dimensions of the crystals, a packing scheme for the peptide is proposed that is consistent with a tetrameric channel.

  • Fujii G, Horvath S, Woodward S, Eiserling F, Eisenberg D. (1992). A molecular model for membrane fusion based on solution studies of an amphiphilic peptide from HIV gp41.Protein Sci.. Nov 1992. 1(11):1454-64. [Abstract]

    The mechanism of protein-mediated membrane fusion and lysis has been investigated by solution-state studies of the effects of peptides on liposomes. A peptide (SI) corresponding to a highly amphiphilic C-terminal segment from the envelope protein (gp41) of the human immunodeficiency virus (HIV) was synthesized and tested for its ability to cause lipid membranes to fuse together (fusion) or to break open (lysis). These effects were compared to those produced by the lytic and fusogenic peptide from bee venom, melittin. Other properties studied included the changes in visible absorbance and mean particle size, and the secondary structure of peptides as judged by CD spectroscopy. Taken together, the observations suggest that protein-mediated membrane fusion is dependent not only on hydrophobic and electrostatic forces but also on the spatial arrangement of the amino acid residues to form an amphiphilic structure that promotes the mixing of the lipids between membranes. A speculative molecular model is proposed for membrane fusion by alpha-helical peptides, and its relationship to the forces involved in protein-membrane interactions is discussed.


  • Lüthy R, McLachlan AD, Eisenberg D. (1991). Secondary structure-based profiles: use of structure-conserving scoring tables in searching protein sequence databases for structural similarities.Proteins. 1991. 10(3):229-39. [Abstract]

    The profile method, for detecting distantly related proteins by sequence comparison, has been extended to incorporate secondary structure information from known X-ray structures. The sequence of a known structure is aligned to sequences of other members of a given folding class. From the known structure, the secondary structure (alpha-helix, beta-strand or “other”) is assigned to each position of the aligned sequences. As in the standard profile method, a position-dependent scoring table, termed a profile, is calculated from the aligned sequences. However, rather than using the standard Dayhoff mutation table in calculating the profile, we use distinct amino acid mutation tables for residues in alpha-helices, beta-strands or other secondary structures to calculate the profile. In addition, we also distinguish between internal and external residues. With this new secondary structure-based profile method, we created a profile for eight-stranded, antiparallel beta barrels of the insecticyanin folding class. It is based on the sequences of retinol-binding protein, insecticyanin and beta-lactoglobulin. Scanning the sequence database with this profile, it was possible to detect the sequence of avidin. The structure of streptavidin is known, and it appears to be distantly related to the antiparallel beta barrels. Also detected is the sequence of complement component C8, which we therefore predict to be a member of this folding class.

  • Hill CP, Yee J, Selsted ME, Eisenberg D. (1991). Crystal structure of defensin HNP-3, an amphiphilic dimer: mechanisms of membrane permeabilization.Science. Mar 1991. 251(5000):1481-5. [Abstract]

    Defensins (molecular weight 3500 to 4000) act in the mammalian immune response by permeabilizing the plasma membranes of a broad spectrum of target organisms, including bacteria, fungi, and enveloped viruses. The high-resolution crystal structure of defensin HNP-3 (1.9 angstrom resolution, R factor 0.19) reveals a dimeric beta sheet that has an architecture very different from other lytic peptides. The dimeric assembly suggests mechanisms by which defensins might bind to and permeabilize the lipid bilayer.

  • Bowie JU, Lüthy R, Eisenberg D. (1991). A method to identify protein sequences that fold into a known three-dimensional structure.Science. Jul 1991. 253(5016):164-70. [Abstract]

    The inverse protein folding problem, the problem of finding which amino acid sequences fold into a known three-dimensional (3D) structure, can be effectively attacked by finding sequences that are most compatible with the environments of the residues in the 3D structure. The environments are described by: (i) the area of the residue buried in the protein and inaccessible to solvent; (ii) the fraction of side-chain area that is covered by polar atoms (O and N); and (iii) the local secondary structure. Examples of this 3D profile method are presented for four families of proteins: the globins, cyclic AMP (adenosine 3′,5′-monophosphate) receptor-like proteins, the periplasmic binding proteins, and the actins. This method is able to detect the structural similarity of the actins and 70- kilodalton heat shock proteins, even though these protein families share no detectable sequence similarity.

  • Fujii G, Choe SH, Bennett MJ, Eisenberg D. (1991). Crystallization of diphtheria toxin.J. Mol. Biol.. Dec 1991. 222(4):861-4. [Abstract]

    Two new crystal forms (forms III and IV) have been grown of diphtheria toxin (DT), which kills susceptible cells by catalyzing the ADP-ribosylation of elongation factor 2, thereby stopping protein synthesis. Forms III and IV diffract to 2.3 A and 2.7 A resolution, respectively. Both forms belong to space group C2; the unit cell parameters for form III are a = 107.3 A, b = 91.7 A, c = 66.3 A and beta = 94.7 degrees and those for form IV are a = 108.3 A, b = 92.3 A, c = 66.1 A and beta = 90.4 degrees. Both forms have one protein chain per asymmetric unit with the dimeric molecule on a twofold axis of symmetry. Form IV is exceptional among all crystal forms of DT in that it can be grown reproducibly. Thus the form IV crystals should yield a crystallographic structure giving insight into the catalytic, receptor-binding and membrane-insertion properties of DT.


  • Eisenberg D, Wesson M. (1990). The most highly amphiphilic alpha-helices include two amino acid segments in human immunodeficiency virus glycoprotein 41.Biopolymers. Jan 1990. 29(1):171-7. [Abstract]

    A search for highly amphiphilic alpha-helices has been made in a data base of protein sequences, using the helical hydrophobic moment as a criterion of amphiphilicity. The protein segments of largest hydrophobic moment have been analyzed. For the segments whose structures are known, they are in fact alpha-helices. Two of the segments having very large hydrophobic moments are from the smaller C-terminal portion of the human immunodeficiency virus (HIV) envelope glycoprotein gp41. Also, among segments having large hydrophobic moments, but not among the most extreme, are lytic peptides such as melittin. Melittin seeks surfaces between polar and apolar phases, including the membrane-water interface. It is conceivable that the gp41 segments of extreme hydrophobic moment may participate in one of the membrane-related functions of the HIV virus.

  • Hill CP, Anderson DH, Wesson L, DeGrado WF, Eisenberg D. (1990). Crystal structure of alpha 1: implications for protein design.Science. Aug 1990. 249(4968):543-6. [Abstract]

    X-ray diffraction shows the structure of a synthetic protein model, formed from noncovalent self-association of a 12-residue peptide and of sulfate ions at low pH. This peptide is a fragment of a 16-residue polypeptide that was designed to form an amphiphilic alpha helix with a ridge of Leu residues along one helical face. By interdigitation of the leucines of four such helices, the design called for self-association into a four-alpha-helical bundle. The crystal structure (2.7 angstrom resolution; R factor = 0.215) reveals a structure more complex than the design, with both a tetramer and a hexamer. The alpha-helical tetramer with leucine interior has more oblique crossing angles than most four-alpha-helical bundles; the hexamer has a globular hydrophobic core of 12 leucine residues and three associated sulfate ions. Computational analysis suggests that the hexameric association is tighter than the tetrameric one. The consistency of the structure with the design is discussed, as well as the divergence.

  • Yamashita MM, Wesson L, Eisenman G, Eisenberg D. (1990). Where metal ions bind in proteins.Proc. Natl. Acad. Sci. U.S.A.. Aug 1990. 87(15):5648-52. [Abstract]

    The environments of metal ions (Li+, Na+, K+, Ag+, Cs+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+) in proteins and other metal-host molecules have been examined. Regardless of the metal and its precise pattern of ligation to the protein, there is a common qualitative feature to the binding site: the metal is ligated by a shell of hydrophilic atomic groups (containing oxygen, nitrogen, or sulfur atoms) and this hydrophilic shell is embedded within a larger shell of hydrophobic atomic groups (containing carbon atoms). That is, metals bind at centers of high hydrophobicity contrast. This qualitative observation can be described analytically by the hydrophobicity contrast function, C, evaluated from the structure. This function is large and positive for a sphere of hydrophilic atomic groups (characterized by atomic solvation parameters, delta sigma, having values less than 0) at the center of a larger sphere of hydrophobic atomic groups (characterized by delta sigma greater than 0). In the 23 metal-binding molecules we have examined, the maximum values of the contrast function lie near to observed metal binding sites. This suggests that the hydrophobicity contrast function may be useful for locating, characterizing, and designing metal binding sites in proteins.

  • Anderson D, Raffioni S, Luporini P, Bradshaw RA, Eisenberg D. (1990). Crystallization of the Euplotes raikovi mating pheromone Er-1.J. Mol. Biol.. Nov 1990. 216(1):1-2. [Abstract]

    A protein mating pheromone Er-1 from the ciliate Euplotes raikovi has been crystallized from (NH4)2SO4 in two forms. Both are suitable for structural studies to at least 2.8 A resolution. Both unit cell sizes are consistent with a tetramer of molecular weight 17,640 in the asymmetric unit.


  • Kantardjieff K, Collier RJ, Eisenberg D. (1989). X-ray grade crystals of the enzymatic fragment of diphtheria toxin.J. Biol. Chem.. Jun 1989. 264(18):10402-4. [Abstract]

    The enzymatic fragment of diphtheria toxin, fragment A (Mr = 21,167), complexed to the dinucleotide adenosine 3′,5′-uridine (ApU), has been crystallized at two different values of pH by hanging drop vapor diffusion. Crystals grown at a pH value of 5.0 (from I) belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 71.2 A, b = 73.0 A, c = 139.8 A and four protomers in the asymmetric unit. Crystals grown at a pH value of 8.1 (form II) belong to the monoclinic space group C2, with unit cell parameters a = 65.2 A, b = 85.6 A, c = 34.6 A, beta = 103.0 degrees and one protomer in the asymmetric unit. Both crystal forms diffract to 2.5 A resolution. The molecular structures of fragment A obtained from these two crystal forms may illuminate the pH-dependent transition of diphtheria toxin during membrane translocation.

  • Rees DC, DeAntonio L, Eisenberg D. (1989). Hydrophobic organization of membrane proteins.Science. Aug 1989. 245(4917):510-3. [Abstract]

    Membrane-exposed residues are more hydrophobic than buried interior residues in the transmembrane regions of the photosynthetic reaction center from Rhodobacter sphaeroides. This hydrophobic organization is opposite to that of water-soluble proteins. The relative polarities of interior and surface residues of membrane and water soluble proteins are not simply reversed, however. The hydrophobicities of interior residues of both membrane and water-soluble proteins are comparable, whereas the bilayer-exposed residues of membrane proteins are more hydrophobic than the interior residues, and the aqueous-exposed residues of water-soluble proteins are more hydrophilic than the interior residues. A method of sequence analysis is described, based on the periodicity of residue replacement in homologous sequences, that extends conclusions derived from the known atomic structure of the reaction center to the more extensive database of putative transmembrane helical sequences.

  • Yamashita MM, Almassy RJ, Janson CA, Cascio D, Eisenberg D. (1989). Refined atomic model of glutamine synthetase at 3.5 A resolution.J. Biol. Chem.. Oct 1989. 264(30):17681-90. [Abstract]

    An atomic model of 43,692 non-hydrogen atoms has been determined for the 12-subunit enzyme glutamine synthetase from Salmonella typhimurium, by methods of x-ray diffraction including restrained least-squares atomic refinement against 65,223 unique reflections. At 3.5 A resolution the crystallographic R-factor (on 2 sigma data) is 25.8%. As reported earlier for the unrefined structure, the 12 subunits are arranged in two layers of six; at the interface of pairs of subunits within each layer, cylindrical active sites are formed by six anti-parallel beta strands contributed by one subunit and two strands by the neighboring subunit. This interpretation of the electron density map has now been supported by comparison with glutamine synthetase from Escherichia coli by the Fourier difference method. Each active site cylinder holds two Mn2+ ions, with each ion having as ligands three protein side chains and two water molecules (one water shared by both metals), as well as a histidyl side chain just beyond liganding distance. The protein ligands to Mn2+ 469 are Glu-131, Glu-212, and Glu-220; those to Mn2+ 470 are Glu-129, His-269, and Glu-357. The two layers of subunits are held together largely by the apolar COOH terminus, a helical thong, which inserts into a hydrophobic pocket formed by two neighboring subunits on the opposite ring. Also between layers, there is a hydrogen-bonded beta sheet interaction, as there is between subunits within a ring, but hydrophobic interactions account for most of the intersubunit stability. The central loop, which extends into the central aqueous channel, is subject to attack by at least five enzymes and is discussed as an enzyme “passive site.”


  • Gribskov M, Homyak M, Edenfield J, Eisenberg D. (1988). Profile scanning for three-dimensional structural patterns in protein sequences.Comput. Appl. Biosci.. Mar 1988. 4(1):61-6. [Abstract]

    Profile analysis measures the similarity between a target sequence and a group of aligned sequences (the probe). The probe sequences are used to produce a position-specific scoring table (the profile) that can be aligned with any sequence (the target) using standard dynamic programming methods. We are developing a library of profiles, each describing a different structural motif. This allows any target sequence to be rapidly scanned for the presence of structural motifs. Levels of significance for the comparison of target sequences with the profile are determined in advance, permitting an objective decision to be made as to whether a protein is likely to possess a structural motif.

  • Silberman H, Eisenberg D, Ryan J, Shofler R, Niland J, Kaptein E, Nicoloff J, Spencer C. (1988). The relation of thyroid indices in the critically ill patient to prognosis and nutritional factors.Surg Gynecol Obstet. Mar 1988. 166(3):223-8. [Abstract]

    Thyroid indices and nutritional assessment values were measured in 73 critically ill euthyroid patients within 48 hours of admission to the medical or surgical intensive care unit. Significantly increased rates of mortality were observed among patients with decreased T3 or T4 levels or elevated T3UR or rT3 values. Alterations in thyroid function tests were associated with changes in certain putative nutritional indices, including serum albumin and transferrin concentrations, triceps skin fold and skin test reactivity. The data indicated that thyroid parameters measured early in the critical phase of illness are predictive of subsequent outcome. The pathogenesis of altered thyroid hormone metabolism is unknown, but nutritional deprivation may be an important contributing factor.

  • Chapman MS, Suh SW, Curmi PM, Cascio D, Smith WW, Eisenberg DS. (1988). Tertiary structure of plant RuBisCO: domains and their contacts.Science. Jul 1988. 241(4861):71-4. [Abstract]

    The three-dimensional structure of ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO), has been determined at 2.6 A resolution. This enzyme initiates photosynthesis by combining carbon dioxide with ribulose bisphosphate to form two molecules of 3-phosphoglycerate. In plants, RuBisCO is built from eight large (L) and eight small (S) polypeptide chains, or subunits. Both S chains and the NH2-terminal domain (N) of L are antiparallel beta, “open-face-sandwich” domains with four-stranded beta sheets and flanking alpha helices. The main domain (B) of L is an alpha/beta barrel containing most of the catalytic residues. The active site is in a pocket at the opening of the barrel that is partly covered by the N domain of a neighboring L chain. The domain contacts of the molecule and its conserved residues are discussed in terms of this structure.

  • Hermanson GG, Eisenberg D, Kincade PW, Wall R. (1988). B29: a member of the immunoglobulin gene superfamily exclusively expressed on beta-lineage cells.Proc. Natl. Acad. Sci. U.S.A.. Sep 1988. 85(18):6890-4. [Abstract]

    A number of the glycoproteins identified on the surfaces of cells of the immune response belong to the immunoglobulin superfamily. We have isolated and characterized cDNA clones and the complete genomic gene encoding a B-cell-specific member of the immunoglobulin superfamily called “B29.” This isolate is expressed at all stages in B-cell development beginning with the earliest precursor B cells undergoing immunoglobulin heavy chain gene diversity region—-joining region gene (DH—-JH) rearrangements. The protein sequence predicted by the B29 coding region contains a leader sequence and a single extracellular immunoglobulin-like domain, followed by a hydrophobic transmembrane segment and a charged intracytoplasmic domain. The immunoglobulin-like domain contains cysteines and other conserved amino acids characteristic of light chain variable and joining regions, but overall the sequence is only distantly related to immunoglobulins. Each of these domains is encoded in separate exons in the B29 gene, in analogy to other members of the immunoglobulin superfamily. The conserved structural features of the immunoglobulin-like domain in the B29 gene product resemble those of other members of the immunoglobulin superfamily involved in cell recognition and adhesion.


  • Eisenberg D, Almassy RJ, Janson CA, Chapman MS, Suh SW, Cascio D, Smith WW. (1987). Some evolutionary relationships of the primary biological catalysts glutamine synthetase and RuBisCO.Cold Spring Harb. Symp. Quant. Biol.. 1987. 52:483-90. [Abstract]
  • Suh SW, Cascio D, Chapman MS, Eisenberg D. (1987). A crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum in the activated state.J. Mol. Biol.. Sep 1987. 197(2):363-5. [Abstract]

    A new crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC from Nicotiana tabacum has been obtained at alkaline pH with polyethylene glycol 8000 in the presence of a non-ionic detergent, beta-octyl glucoside. The crystals are grown at room temperature by the hanging-drop vapor diffusion technique from a protein solution containing enzyme complexed with CO2, Mg2+, and the transition state analog 2-C-carboxy-D-arabinitol-1,5-bisphosphate. The crystals belong to the the space group P3(1)21 (or P3(2)21) with the cell parameters a = 204.6 A, and c = 117.4 A (1 A = 0.1 nm). The asymmetric unit contains half (L4S4: L, large subunit, 53,000 Mr; S, small subunit, 15,000 Mr) of a hexadecameric molecule (L8S8, 540,000 Mr). The crystals diffract to at least 2.6 A Bragg spacing and are suitable for X-ray structure determination.


  • Eisenberg D, Wilcox W, Eshita SM, Pryciak PM, Ho SP, DeGrado WF. (1986). The design, synthesis, and crystallization of an alpha-helical peptide.Proteins. Sep 1986. 1(1):16-22. [Abstract]

    Twelve- and sixteen-residue peptides have been designed to form tetrameric alpha-helical bundles. Both peptides are capable of folding into amphiphilic alpha-helices, with leucyl residues along one face and glutamyl and lysyl residues along the opposite face. Four such amphiphilic alpha-helices are capable of forming a noncovalently bonded tetramer. Neighboring helices run in antiparallel directions in the design, so that the complex has 222 symmetry. In the designed tetramer, the leucyl side chains interdigitate in the center in a hydrophobic interaction, and charged side chains are exposed to the solvent. The designed 12-mer (ALPHA-1) has been synthesized, and it forms helical aggregates in aqueous solution as judged by circular dichroic spectroscopy. It has also been crystallized and characterized by x-ray diffraction. The crystal symmetry is compatible with (but does not prove) the design. The design can be extended to a four-alpha-helical bundle formed from a single polypeptide by adding three peptide linkers.


  • Baker TS, Suh SW, Eisenberg D. (1977). Structure of ribulose-1,5-bisphosphate carboxylase-oxygenase: Form III crystals.Proc. Natl. Acad. Sci. U.S.A.. Mar 1977. 74(3):1037-41. [Abstract]

    A new crystal form (III) of tobacco leaf ribulosebisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC] has been grown by dialysis procedures, and is suitable for structural studies at near atomic resolution. The crystals exhibit birefringence, grow as pseudo-regular rhombic dodecahedrons, and belong to the tetragonal space group P4(2)2(1)2 with a = b = 149 A, c = 138 A, and V = 3.04 x 10(6) A(3). Each unit cell contains two molecules, with two large and two small subunits per asymmetric unit. At low resolution (>10 A) the crystal structure is body centered belonging to space group 1422 with one large/small pair in the asymmetric unit. Thus, at low resolution the molecular symmetry is D(4), the highest possible symmetry for an oligomer of stoichiometry large(8)small(8). Form III crystals may be identical to crystalline inclusions found in chloroplasts.

  • Baker TS, Suh SW, Eisenberg D. (1977). Structure of ribulose-1,5-bisphosphate carboxylase-oxygenase: Form III crystals.Proc. Natl. Acad. Sci. U.S.A.. Mar 1977. 74(3):1037-41. [Abstract]

    A new crystal form (III) of tobacco leaf ribulosebisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC] has been grown by dialysis procedures, and is suitable for structural studies at near atomic resolution. The crystals exhibit birefringence, grow as pseudo-regular rhombic dodecahedrons, and belong to the tetragonal space group P4(2)2(1)2 with a = b = 149 A, c = 138 A, and V = 3.04 x 10(6) A(3). Each unit cell contains two molecules, with two large and two small subunits per asymmetric unit. At low resolution (>10 A) the crystal structure is body centered belonging to space group 1422 with one large/small pair in the asymmetric unit. Thus, at low resolution the molecular symmetry is D(4), the highest possible symmetry for an oligomer of stoichiometry large(8)small(8). Form III crystals may be identical to crystalline inclusions found in chloroplasts.

  • Baker TS, Eisenberg D, Eiserling F. (1977). Ribulose bisphosphate carboxylase: a two-layered, square-shaped molecule of symmetry 422.Science. Apr 1977. 196(4287):293-5. [Abstract]

    Electron micrographs and x-ray diffraction patterns of crystals of ribulose bisphosphate carboxylase, probably the most abundant protein on earth, have provided new details of the arrangement of subunits. The eight large subunits and eight small subunits are clustered in two layers, perpendicular to a fourfold axis of symmetry. Viewed down the fourfold axis, the molecule is square-shaped.



  • Baker TS, Eisenberg D, Eiserling FA, Weissman L. (1975). The structure of form I crystals of D-ribulose-1,5-diphosphate carboxylase.J. Mol. Biol.. Feb 1975. 91(4):391-9. [Abstract]
  • Frey TG, Eisenberg D, Eiserling FA. (1975). Glutamine synthetase forms three- and seven-stranded helical cables.Proc. Natl. Acad. Sci. U.S.A.. Sep 1975. 72(9):3402-6. [Abstract]

    When cobaltous ion is bound to glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC], the two-layered hexagonal molecules polymerize face-to-face, to form long strands. The strands then wind round each other to form three- and seven-stranded cables. The structures of these cables are not immediately evident from electron micrographs because of the confusing superposition of front and back portions of the cables. But optical diffraction and filtering by the procedure of Klug and DeRosier leads to interpretable images of the cables. Because a micrograph of the seven-stranded cable contains 24 views of the glutamine synthetase molecule, it is possible to reconstruct the three-dimensional electron density of a cable and its constituent molecules at a resolution of 30–50 A. This reconstruction confirms that the symmetry of a glutamine synthetase molecule is D6. It suggests that the single subunit is an oblate ellipsoid with its minor axis (about 48 A) roughly parallel to the 6-fold axis of the molecule and its major axis (about 63 A) perpendicular to the 6-fold axis of the molecule. The subunits of the two hexagonal layers of a molecule are eclipsed. Neighboring molecules along a strand also have their hexagonal faces together, but they are rotated about the strand axis by about 7 degrees with respect to one another, rather than being eclipsed. Six outer strands are coiled about a straight central strand, and each forms identical contacts with the central strand. Moreover, these contacts between central and outer strands are apparently similar to the contacts between neighboring outer strands.

  • Scanlon WJ, Eisenberg D. (1975). Solvation of crystalline proteins: theory and its application to available data.J. Mol. Biol.. Nov 1975. 98(3):485-502. [Abstract]



  • Heidner EG, Weber BH, Eisenberg D. (1971). Subunit structure of aldolase.Science. Feb 1971. 171(3972):677-9. [Abstract]

    A new crystal form of rabbit muscle aldolase shows that the molecule has 222 symmetry to at least 4-angstrom resolution, and hence that the gross conformation of the four subunits is the same. Comparison of the new form with a previously reported form establishes the number of molecules per unit cell, n, in the older form. For an independent check, the “crystal-volume and protein-content method” was developed to determine n without directly measuring the water content of the crystals.

  • Dickerson RE, Takano T, Eisenberg D, Kallai OB, Samson L, Cooper A, Margoliash E. (1971). Ferricytochrome c. I. General features of the horse and bonito proteins at 2.8 A resolution.J. Biol. Chem.. Mar 1971. 246(5):1511-35. [Abstract]




  • Rokito AS, Zuckerman JD, Shaari JM, Eisenberg DP, Cuomo F, Gallagher MA. (Other). A comparison of nonoperative and operative treatment of type II distal clavicle fractures.Bull Hosp Jt Dis. . 61(1-2):32-9. [Abstract]

    A retrospective study was performed to compare nonoperative and operative treatments of Type II distal clavicle fractures. From a total of 30 diagnosed patients, 16 were identified as receiving nonoperative treatment and 14 open reduction and coracoclavicular stabilization. The average follow-up was 53.5 months for the nonoperative group and 59.8 months for the operative group. All patients were evaluated postoperatively for pain, range of motion, function, and fracture healing as well as for isokinetic strength. Fractures treated surgically achieved union within six to ten weeks. Nonoperative treatment resulted in seven nonunions. There were no significant differences between the two groups in the mean UCLA, Constant, and ASES scores. Nonunion had no significant effect on functional outcome or strength. This study suggests that Type II distal clavicle fractures can be successfully managed nonoperatively. The high incidence of nonunion does not impede a clinical outcome comparable to that achieved by surgical treatment.