Biomedical subjects

Miriam Eisenstein

Publications and source records attributed to Miriam Eisenstein.

At least 19 recordsLinked to original sources

PubMed2006

Inherent limitations in protein-protein docking procedures.

MOTIVATION: The limited success rate of protein-protein docking procedures is generally attributed to structure differences between the bound and unbound states of the molecules. Herein we analyze a large dataset of protein-protein docking results and identify additional parameters that affect the performance of docking procedures. RESULTS: We find that the distinction between nearly correct models (NCMs) and decoys depends on the size of the interface to be predicted thus setting a limit to the prediction ability of docking procedures, particularly those in which the geometric complementarity descriptor is dominant. The geometric complementarity score in grid-based docking carries a large statistical error which further reduces the distinction between NCMs and decoys. We propose a method for correcting the statistical error and show that the distinction is improved when the docking models are ranked by statistically equivalent scores. AVAILABILITY: MolFit can be downloaded from our website http://www.weizmann.ac.il/Chemical_Research_Support/molfit. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Algorithms↗

PubMed2006

Vimentin binding to phosphorylated Erk sterically hinders enzymatic dephosphorylation of the kinase.

Cleavage fragments of de novo synthesized vimentin were recently reported to interact with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling complex retrogradely transported on importins and dynein. Here we clarify the structural basis for this interaction, which explains how pErk is protected from dephosphorylation while bound to vimentin. Pull-down and ELISA experiments revealed robust calcium-dependent binding of pErk to the second coiled-coil domain of vimentin, with observed affinities of binding increasing from 180 nM at 0.1 microM calcium to 15 nM at 10 microM calcium. In contrast there was little or no binding of non-phosphorylated Erk to vimentin under these conditions. Geometric and electrostatic complementarity docking generated a number of solutions wherein vimentin binding to pErk occludes the lip containing the phosphorylated residues in the kinase. Binding competition experiments with Erk peptides confirmed a solution in which vimentin covers the phosphorylation lip in pErk, interacting with residues above and below the lip. The same peptides inhibited pErk binding to the dynein complex in sciatic nerve axoplasm, and interfered with protection from phosphatases by vimentin. Thus, a soluble intermediate filament fragment interacts with a signaling kinase and protects it from dephosphorylation by calcium-dependent steric hindrance.

Animals↗

PubMed2006

Identification of novel glycogen synthase kinase-3beta substrate-interacting residues suggests a common mechanism for substrate recognition.

Substrate recognition and specificity are essential for the reliability and fidelity of protein kinase function. GSK-3 has a unique substrate specificity that requires prior phosphorylation of its substrates. However, how the enzyme selects its phosphorylated substrates is unknown. Here, we combined in silico modeling with mutagenesis and biological studies to identify GSK-3-substrate interaction sites located within its binding cleft. Protein-protein docking of GSK-3beta and the phosphorylated cAMP responsive element binding protein (pCREB) (using the available experimentally determined structures), identified Phe67, Gln89, and Asn95 of GSK-3beta as putative binding sites interacting with the CREB phosphorylation motif. Mutations of these residues to alanine impaired GSK-3beta phosphorylation of several substrates, without abrogating its autocatalytic activity. Subsequently, expression of the GSK-3beta mutants in cells resulted in decreased phosphorylation of substrates CREB, IRS-1, and beta-catenin, and prevented their suppression of glycogen synthase activity as compared with cells expressing the wild-type GSK-3beta. Our studies provide important additional understanding of how GSK-3beta recognizes its substrates: In addition to prior phosphorylation typically required in GSK-3 substrates, substrate recognition involves interactions with GSK-3beta residues: Phe67, Gln89, and Asn95, which confer a common basis for substrate binding and selectivity, yet allow for substrate diversity.

Amino Acid Sequence↗

PubMed2006

Hsp90 recognizes a common surface on client kinases.

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.

Amino Acid Sequence↗

PubMed2006

It depends on the hinge: a structure-functional analysis of galectin-8, a tandem-repeat type lectin.

Galectin-8, a member of the galectin family of mammalian lectins, is made of two carbohydrate-recognition domains (CRDs), joined by a "hinge" region. Ligation of integrins by galectin-8 induces a distinct cytoskeletal organization, associated with activation of the extracellular-regulated kinase (ERK) and phosphatidylinositol 3-kinase signaling cascades. We show that these properties of galectin-8 are mediated by the concerted action of its two CRDs and involve both protein-sugar and protein-protein interactions. Accordingly, the isolated N- or C-CRD domains of galectin-8 or galectin-8 mutated at selected residues implicated in sugar binding (E251Q; W85Y, W248Y, W[85,248]Y) exhibited reduced sugar binding, which was accompanied by severe impairment in the capacity of these mutants to promote the adhesive, spreading, and signaling functions of galectin-8. Other mutations that did not impair sugar binding (e.g. E88Q) still impeded the signaling and cell-adherence functions of galectin-8. Deletion of the "hinge" region similarly impaired the biological effects of galectin-8. These results provide evidence that cooperative interactions between the two CRDs and the "hinge" domain are required for the proper functioning of galectin-8.

Animals↗

PubMed2006

Anatomy of T cell autoimmunity to myelin oligodendrocyte glycoprotein (MOG): prime role of MOG44F in selection and control of MOG-reactive T cells in H-2b mice.

Myelin oligodendrocyte glycoprotein (MOG) is an important myelin target antigen, and MOG-induced EAE is now a widely used model for multiple sclerosis. Clonal dissection revealed that MOG-induced EAE in H-2(b) mice is associated with activation of an unexpectedly large number of T cell clones reactive against the encephalitogenic epitope MOG35-55. These clones expressed extremely diverse TCR with no obvious CDR3alpha/CDR3beta motif(s). Despite extensive TCR diversity, the cells required MOG40-48 as their common core epitope and shared MOG44F as their major TCR contact. Fine epitope-specificity analysis with progressively truncated peptides suggested that the extensive TCR heterogeneity is mostly related to differential recognition of multiple overlapping epitopes nested within MOG37-52, each comprised of a MOG40-48 core flanked at the N- and/or the C-terminus by a variable number of residues important for interaction with different TCR. Abrogation of both the encephalitogenic potential of MOG and T cell reactivity against MOG by a single mutation (MOG44F/MOG44A), together with effective down-regulation of MOG-induced EAE by MOG37-44A-52, confirmed in vivo the primary role for MOG44F in the selection/activation of MOG-reactive T cells. We suggest that such a highly focused T cell autoreactivity could be a selective force that offsets the extensive TCR diversity to facilitate a more "centralized control" of pathogenic MOG-related T cell autoimmunity.

Amino Acid Substitution↗

PubMed2005

Looking at enzymes from the inside out: the proximity of catalytic residues to the molecular centroid can be used for detection of active sites and enzyme-ligand interfaces.

Analysis of the distances of the exposed residues in 175 enzymes from the centroids of the molecules indicates that catalytic residues are very often found among the 5% of residues closest to the enzyme centroid. This property of catalytic residues is implemented in a new prediction algorithm (named EnSite) for locating the active sites of enzymes and in a new scheme for re-ranking enzyme-ligand docking solutions. EnSite examines only 5% of the molecular surface (represented by surface dots) that is closest to the centroid, identifying continuous surface segments and ranking them by their area size. EnSite ranks the correct prediction 1-4 in 97% of the cases in a dataset of 65 monomeric enzymes (rank 1 for 89% of the cases) and in 86% of the cases in a dataset of 176 monomeric and multimeric enzymes from all six top-level enzyme classifications (rank 1 in 74% of the cases). Importantly, identification of buried or flat active sites is straightforward because EnSite "looks" at the molecular surface from the inside out. Detailed examination of the results indicates that the proximity of the catalytic residues to the centroid is a property of the functional unit, defined as the assembly of domains or chains that form the active site (in most cases the functional unit corresponds to a single whole polypeptide chain). Using the functional unit in the prediction further improves the results. The new property of active sites is also used for re-evaluating enzyme-inhibitor unbound docking results. Sorting the docking solutions by the distance of the interface to the centroid of the enzyme improves remarkably the ranks of nearly correct solutions compared to ranks based on geometric-electrostatic-hydrophobic complementarity scores.

Algorithms↗

PubMed2005

Modeling oligomers with Cn or Dn symmetry: application to CAPRI target 10.

The abundance of oligomeric proteins makes them a frequent target for structure prediction. However, homologous proteins sometimes adopt different oligomerization states, rendering the prediction of structures of whole oligomers beyond the scope of comparative modeling. This obstacle can be overcome by combining comparative modeling of the single subunit of an oligomer with docking techniques, designed for predicting subunit-subunit interfaces. We present here algorithms for predicting the structures of homo-oligomers with C(n) or D(n) (n > 2) symmetry. The prediction procedure includes a symmetry-restricted docking step followed by a C(n) or D(n) oligomer-forming step, in which the dimers from the docking step are assembled to oligomers. The procedure is applied to each of the crystallographically independent subunits in 8 C(n) and 3 D(n) oligomers, producing very accurate predictions. It is further applied to a single monomer of the tick-borne encephalitis virus coat protein E (Target 10 of the CAPRI experiment). The predicted trimer ranked 30, obtained via rigid-body geometric-hydrophobic docking followed by C(n) oligomer formation, is very similar to the experimentally observed trimer formed by domain II of this protein. Furthermore, the predicted trimer formed from the separated domain I is also close to the experimental structure.

Algorithms↗

PubMed2005

Detection and reduction of evolutionary noise in correlated mutation analysis.

Direct or indirect inter-residue interactions in proteins are often reflected by mutations at one site that compensate for mutations at another site. Various bioinformatic methods have been developed for detecting such correlated mutations in order to obtain information about intra- and inter-protein interactions. Here, we show by carrying out a correlated mutation analysis for non-interacting proteins that the signal due to inter-residue interactions is of similar magnitude to the 'noise' that arises from other evolutionary processes related to common ancestry. A new method for detecting correlated mutations is presented that reduces this evolutionary noise by taking into account evolutionary distances in the protein family. It is shown that this method yields better signal-to-noise ratios and, therefore, can much better resolve, for example, correlated mutations that reflect true inter-residue interactions.

Computational Biology↗

PubMed2005

The effect of resolution-dependent global shape modifications on rigid-body protein-protein docking.

Docking unbound molecules presents a challenge in the case where no prior biological or bioinformatic knowledge exists. This is mainly due to differences between the structures of the molecules when in a complex and in the free state. Presumably, these differences interfere with the ability of protein-protein docking algorithms, which rely on a dominant shape descriptor, to identify the correct solution and rank it higher than false solutions. In this study we verify the notion that small discords in the molecular fit can be eliminated by using appropriately designed low-resolution shape descriptors, thereby improving the docking results. We exploit the inherent gradual resolution dependency of Fourier transforms and formulate a resolution-dependent shape descriptor by truncating selected Fourier transform terms. Thus, different levels of shape modification are attained, affecting the degree of detail in the depiction of the molecular surface. We applied the modified descriptor to a selection of 23 protein-protein systems, using the unbound structures where possible. The docking results obtained with the new geometric descriptor were considerably superior to former results, improving the ranks of nearly correct solutions for 17 systems. Unification of the results of scans in which different resolutions were employed further improved the ranks of nearly correct solutions to less than 100 for 12 of the 23 systems and less than 300 for 20 systems. The new geometric descriptor can be combined with other descriptors, which typify the electrostatic or hydrophobic character of the molecular surface, and with external experimental or bioinformatic data.

Binding Sites↗

PubMed2005

Experimental and computational characterization of the dimerization of the PTS-regulation domains of BglG from Escherichia coli.

BglG and LicT are transcriptional antiterminators from Escherichia coli and Bacillus subtilis, respectively, that control the expression of genes and operons involved in transport and catabolism of carbohydrates. Both proteins contain a duplicate conserved domain, the PTS-regulation domain (PRD), and they are regulated by phosphorylation on specific, highly conserved histidine residues located in the PRDs. However, despite their similar function and the high sequence identity, experimental evidence implies different modes of regulation. Thus, BglG must be de-phosphorylated on PRD2 in order to form active dimers, whereas activation of LicT requires de-phosphorylation on PRD1 and phosphorylation on PRD2. Here we address two goals. First, we test in vivo and in silico the effect of point mutations in the PRDs of BglG on the PRD-PRD dimerization. Second, we explore computationally the effect of histidine phosphorylation on PRD dimerization in BglG and LicT. We find excellent correspondence between the experimental and computational measures of the influence of mutations on PRD dimerization in BglG. This establishes that the geometric-electrostatic complementarity scores computed with the program MolFit provide a good measure of the effects of mutations in this system. In addition, it indicates that the dimerization mode of the separately expressed PRDs of BglG is similar to the dimers formed by activated LicT. The computations also show that phosphorylation of the histidine residues in PRD1 of either BglG or LicT leads to a strong electrostatic repulsion. Conversely, the phosphorylation of one histidine residue in PRD2 of LicT leads to improved electrostatic complementarity at the PRD2-PRD2 interface, whereas the corresponding phosphorylation in BglG has negligible contribution. This different conduct may be attributed to a single replacement in the sequence of PRD2 in BglG compared to LicT, Ala262 versus Asp261, respectively.

Bacterial Proteins↗

PubMed2005

The structure and synthetic capabilities of a catalytic peptide formed by substrate-directed mechanism--implications to prebiotic catalysis.

Previously, we have shown that a small substrate may serve as a template in the formation of a specific catalytic peptide, a phenomenon which might have had a major role in prebiotic synthesis of peptide catalysts. This was demonstrated experimentally by the formation of a catalytic metallo-dipeptide, Cys2-Fe2+, around o-nitrophenyl beta-D-galactopyranoside (ONPG), by dicyandiamide (DCDA)-assisted condensation under aqueous conditions. This dipeptide was capable of hydrolyzing ONPG at a specific activity lower only 1000 fold than that of beta galactosidase. In the present paper we use molecular modeling techniques to elucidate the structure of this catalyst and its complex with the substrate and propose a putative mechanism for the catalyst formation and its mode of action as a "mini enzyme". This model suggests that interaction of Fe2+ ion with ONPG oxygens and with two cysteine SH groups promotes the specific formation of the Cys2-Fe2+ catalyst. Similarly, the interaction of the catalyst with ONPG is mediated by its Fe2+ with the substrate oxygens, leading to its hydrolysis. In addition, immobilized forms of the catalyst were synthesized on two carriers--Eupergit C and amino glass beads. These preparations were capable of catalyzing the formation of ONPG from beta-D-galactose and o-nitrophenol (ONP) under anhydrous conditions. The ability of the catalyst to synthesize the substrate that mediates its own formation creates an autocatalytic cycle where ONPG catalyzes the formation of a catalyst which, in turn, catalyzes ONPG formation. Such autocatalytic cycle can only operate by switching between high and low water activity conditions, such as in tidal pools cycling between wet and dry environments. Implications of the substrate-dependent formation of catalytically active peptides to prebiotic processes are discussed.

Catalysis↗

PubMed2004

The olfactory receptor universe--from whole genome analysis to structure and evolution.

Olfactory receptors (ORs) constitute the largest gene-family in the vertebrate genome. We have attempted to provide a comprehensive view of the OR universe through diverse tools of bioinformatics and computational biology. Among others, we have constructed the Human Olfactory Receptor Data Exploratorium (HORDE, http://bioportal.weizmann.ac.il/HORDE/) as a free online resource, which integrates information on ORs from different species. We studied the genomic organization of 853 human ORs and divided the repertoire into 135 clusters, accessible through our new cluster viewer feature. An analysis of intact and pseudogenized ORs in different clusters, as well as of OR expression patterns, is provided, relevant to OR transcription control. Coding single nucleotide polymorphisms were integrated; these are to be used for genotype-phenotype correlation studies. HORDE allows a unique opportunity for discerning protein structural and functional information of the individual OR proteins. By applying novel data analysis strategies to the >3000 OR genes of mouse, dog and human within HORDE, we have generated a set of refined rhodopsin-based homology models for ORs. For model improvement, we employed a novel analysis of specific positions along the seven transmembrane helices at which prolines generate helix-breaking kinks. The model shows family-specific structural features, including idiosyncratic kink patterns, which lead to significant differences in the inferred odorant binding site structure. Such analyses form a basis for a comprehensive sequence-based classification of OR proteins in terms of potential odorant binding specificities.

Amino Acid Sequence↗

PubMed2004

The myelin-associated oligodendrocytic basic protein region MOBP15-36 encompasses the immunodominant major encephalitogenic epitope(s) for SJL/J mice and predicted epitope(s) for multiple sclerosis-associated HLA-DRB1*1501.

Autoimmune response to the myelin-associated oligodendrocytic basic protein (MOBP), a CNS-specific myelin constituent, was recently suggested to play a role in the pathogenesis of multiple sclerosis (MS). The pathogenic autoimmune response to MOBP and the associated pathology in the CNS have not yet been fully investigated. In this study, we have characterized the clinical manifestations, pathology, T cell epitope-specificity, and TCRs associated with experimental autoimmune encephalomyelitis (EAE) induced in SJL/J mice with recombinant mouse MOBP (long isoform, 170 aa). Analysis of encephalitogenic MOBP-reactive T cells for reactivity to overlapping MOBP peptides defined MOBP15-36 as their major immunodominant epitope. Accordingly, MOBP15-36 was demonstrated to be the major encephalitogenic MOBP epitope for SJL/J mice, inducing severe/chronic clinical EAE associated with intense perivascular and parenchymal infiltrations, widespread demyelination, axonal loss, and remarkable optic neuritis. Molecular modeling of the interaction of I-A(s) with MOBP15-36, together with analysis of the MOBP15-36-specific T cell response to truncated peptides, suggests MOBP20-28 as the core sequence for I-A(s)-restricted recognition of the encephalitogenic region MOBP15-36. Although highly focused in their epitope specificity, the encephalitogenic MOBP-reactive T cells displayed a widespread usage of TCR Vbeta genes. These results would therefore favor epitope-directed, rather than TCR-targeted, approaches to therapy of MOBP-associated pathogenic autoimmunity. Localization by molecular modeling of a potential HLA-DRB1*1501-associated MOBP epitope within the encephalitogenic MOBP15-36 sequence suggests the potential relevance of T cell reactivity against MOBP15-36 to MS. The reactivity to MOBP15-36 detected in MS shown here and in another study further emphasizes the potential significance of this epitope for MS.

Animals↗

PubMed2004

Hydrophobic complementarity in protein-protein docking.

Formation of hydrophobic contacts across a newly formed interface is energetically favorable. Based on this observation we developed a geometric-hydrophobic docking algorithm that estimates quantitatively the hydrophobic complementarity at protein-protein interfaces. Each molecule to be docked is represented as a grid of complex numbers, storing information regarding the shape of the molecule in the real part and information regarding the hydropathy of the surface in the imaginary part. The grid representations are correlated using fast Fourier transformations. The algorithm is used to compare the extent of hydrophobic complementarity in oligomers (represented by D2 tetramers) and in hetero-dimers of soluble proteins (complexes). We also test the implication of hydrophobic complementarity in distinguishing correct from false docking solutions. We find that hydrophobic complementarity at the interface exists in oligomers and in complexes, and in both groups the extent of such complementarity depends on the size of the interface. Thus, the non-polar portions of large interfaces are more often juxtaposed than non-polar portions of small interfaces. Next we find that hydrophobic complementarity helps to point out correct docking solutions. In oligomers it significantly improves the ranks of nearly correct reassembled and modeled tetramers. Combining geometric, electrostatic and hydrophobic complementarity for complexes gives excellent results, ranking a nearly correct solution < 10 for 5 of 23 tested systems, < 100 for 8 systems and < 1000 for 19 systems.

Algorithms↗

PubMed2004

Antibody recognition of chiral surfaces. Structural models of antibody complexes with leucine-leucine-tyrosine crystal surfaces.

Molecular models are built of the recognition domains of two antibodies, which are raised and selected against crystals of (L)leucine-(L)leucine-(L)tyrosine. The model of one antibody, which is stereo- and enantioselective, reveals astounding chemical and structural complementarity to the recognized crystal surface. The enantioselective binding of this antibody is explained by the significantly fewer chemical interactions arising in the complex, after docking of the antibody to the (D)Leu-(D)Leu-(D)Tyr crystal face, relative to its enantiomer, the (L)Leu-(L)Leu-(L)Tyr crystal face. The modeling and docking of the second antibody, which is poorly stereoselective and is not enantioselective, indicates that binding is based on electrostatic interactions. The docking models of the antibody-crystal complexes provide a rationale for the experimental results while demonstrating the power of modeling techniques to meet the challenge of describing antibody-antigen interactions in detail.

Amino Acid Sequence↗

PubMed2004

Monoclonal antibody to a DNA-binding domain of p53 mimics charge structure of DNA: anti-idiotypes to the anti-p53 antibody are anti-DNA.

Antibodies to DNA are important markers of various autoimmune diseases and can be pathogenic; however, their generation is not understood. We previously reported that anti-DNA antibodies could be induced in mice by idiotypic immunization to PAb-421, an antibody to a DNA-binding domain of p53. We now report that two monoclonal antibodies of moderate affinity (K(D) asymptotically equal to 10(-7)), raised from PAb-421-immunized mice, specifically recognized both PAb-421 and DNA. These antibodies feature multiple arginine residues in the antigen-binding site, a unique characteristic of disease-associated anti-DNA antibodies; nevertheless, these anti-DNA antibodies show specific complementarity to PAb-421 by competing with p53 for PAb-421 binding and recognize defined oligonucleotides with a specificity similar to that of p53. To study the structural basis for the cross-recognition of PAb-421 and DNA by the anti-DNA antibodies, we constructed computer models (fine-tuned by protein-protein docking) of PAb-421 and one of the monoclonal anti-DNA antibodies. The modeled structures manifested structural complementarity. Most notably, the modeled structure of PAb-421 resembled the structure of DNA by the positions of negatively charged groups and aromatic side chains. Thus, a protein molecule may mimic the structure of DNA and the elusive generation of anti-DNA antibodies could be explained by idiotypic immunity to a DNA-binding protein, like p53.

Amino Acid Sequence↗

PubMed2004

On proteins, grids, correlations, and docking.

The activity of a living cell can be portrayed as a network of interactions involving proteins and nucleic acids that transfer biological information. Intervention in cellular processes requires thorough understanding of the interactions between the molecules, which can be provided by docking techniques. Docking methods attempt to predict the structures of complexes given the structures of the component molecules. We focus hereby on protein-protein docking procedures that employ grid representations of the molecules, and use correlation for searching the solution space and evaluating putative complexes. Geometric surface complementarity is the dominant descriptor in docking. Inclusion of electrostatics often improves the results of geometric docking for soluble proteins, whereas hydrophobic complementarity is more important in construction of oligomers. Using binding-site information in the scan or as a filter helps to identify and up-rank nearly correct solutions.

Binding Sites↗