Parameterization and classification of the protein universe via geometric techniques

We present a scheme for the classification of 3487 non-redundant protein structures into 1207 non-hierarchical clusters by using recurring structural patterns of three to six amino acids as keys of classification. This results in several signature patterns, which seem to decide membership of a protein in a functional category. The patterns provide clues to the key residues involved in functional sites as well as in protein-protein interaction. The discovered patterns include a "glutamate double bridge" of superoxide dismutase, the functional interface of the serine protease and inhibitor, interface of homo/hetero dimers, and functional sites of several enzyme families. We use geometric invariants to decide superimposability of structural patterns. This allows the parameterization of patterns and discovery of recurring patterns via clustering. The geometric invariant-based approach eliminates the computationally explosive step of pair-wise comparison of structures. The results provide a vast resource for the biologists for experimental validation of the proposed functional sites, and for the design of synthetic enzymes, inhibitors and drugs.     

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).     

Crystal structure of calcium-free human sorcin: A member of the penta-EF-hand protein family

Sorcin is a 22 kD calcium-binding protein that is found in a wide variety of cell types, such as heart, muscle, brain and adrenal medulla. It belongs to the penta-EF-hand (PEF) protein family, which contains five EF-hand motifs that associate with membranes in a calcium-dependent manner. Prototypic members of this family are the calcium-binding domains of calpain, such as calpain dVI. Full-length human sorcin has been crystallized in the absence of calcium and the structure determined at 2.2 A resolution. Apart from an extended N-terminal portion, the sorcin molecule has a globular shape. The C-terminal domain is predominantly alpha-helical, containing eight alpha-helices and connecting loops incorporating five EF hands. Sorcin forms dimers through the association of the unpaired EF5, confirming this as the mode of association in the dimerization of PEF proteins. Comparison with calpain dVI reveals that the general folds of the individual EF-hand motifs are conserved, especially that of EF1, the novel EF-hand motif characteristic of the family. Detailed structural comparisons of sorcin with other members of PEF indicate that the EF-hand pair EF1-EF2 is likely to correspond to the two physiologically relevant calcium-binding sites and that the calcium-induced conformational change may be modest and localized within this pair of EF-hands. Overall, the results derived from the structural observations support the view that, in sorcin, calcium signaling takes place through the first pair of EF-hands.     

Structure motif discovery and mining the PDB

MOTIVATION: Many of the most interesting functional and evolutionary relationships among proteins are so ancient that they cannot be reliably detected through sequence analysis and are apparent only through a comparison of the tertiary structures. The conserved features can often be described as structural motifs consisting of a few single residues or Secondary Structure (SS) elements. Confidence in such motifs is greatly boosted when they are found in more than a pair of proteins. RESULTS: We describe an algorithm for the automatic discovery of recurring patterns in protein structures. The patterns consist of individual residues having a defined order along the protein's backbone that come close together in the structure and whose spatial conformations are similar. The residues in a pattern need not be close in the protein's sequence. The work described in this paper builds on an earlier reported algorithm for motif discovery. This paper describes a significant improvement of the algorithm which makes it very efficient. The improved efficiency allows us to use it for doing unsupervised learning of patterns occurring in small subsets in a large set of structures, a non-redundant subset of the Protein Data Bank (PDB) database of all known protein structures.     

Mushroom lectins: Current status and future perspectives

Lectins are nonimmune proteins or glycoproteins that bind specifically to cell surface carbohydrates, culminating in cell agglutination. These are known to play key roles in host defense system and also in metastasis. Many new sources have been explored for the occurrence of lectins during the last few years. Numerous novel lectins with unique specificities and exploitable properties have been discovered. Mushrooms have attracted a number of researchers in food and pharmaceuticals. Many species have long been used in traditional Chinese medicines or functional foods in Japan and other Asian countries. A number of bioactive constituents have been isolated from mushrooms including polysaccharides, polysaccharopeptides, polysaccharide–protein complexes, proteases, ribonucleases, ribosome inactivating proteins, antifungal proteins, immunomodulatory proteins, enzymes, lectins, etc. Mushroom lectins are endowed with mitogenic, antiproliferative, antitumor, antiviral, and immunestimulating potential. In this review, an attempt has been made to collate the information on mushroom lectins, their blood group and sugar specificities, with an emphasis on their biomedical potential and future perspectives.     

Recurrent use of evolutionary importance for functional annotation of proteins based on local structural similarity

The annotation of protein function has not kept pace with the exponential growth of raw sequence and structure data. An emerging solution to this problem is to identify 3D motifs or templates in protein structures that are necessary and sufficient determinants of function. Here, we demonstrate the recurrent use of evolutionary trace information to construct such 3D templates for enzymes, search for them in other structures, and distinguish true from spurious matches. Serine protease templates built from evolutionarily important residues distinguish between proteases and other proteins nearly as well as the classic Ser-His-Asp catalytic triad. In 53 enzymes spanning 33 distinct functions, an automated pipeline identifies functionally related proteins with an average positive predictive power of 62%, including correct matches to proteins with the same function but with low sequence identity (the average identity for some templates is only 17%). Although these template building, searching, and match classification strategies are not yet optimized, their sequential implementation demonstrates a functional annotation pipeline which does not require experimental information, but only local molecular mimicry among a small number of evolutionarily important residues.     

A new bioinformatic approach to detect common 3D sites in protein structures

An innovative bioinformatic method has been designed and implemented to detect similar three-dimensional (3D) sites in proteins. This approach allows the comparison of protein structures or substructures and detects local spatial similarities: this method is completely independent from the amino acid sequence and from the backbone structure. In contrast to already existing tools, the basis for this method is a representation of the protein structure by a set of stereochemical groups that are defined independently from the notion of amino acid. An efficient heuristic for finding similarities that uses graphs of triangles of chemical groups to represent the protein structures has been developed. The implementation of this heuristic constitutes a software named SuMo (Surfing the Molecules), which allows the dynamic definition of chemical groups, the selection of sites in the proteins, and the management and screening of databases. To show the relevance of this approach, we focused on two extreme examples illustrating convergent and divergent evolution. In two unrelated serine proteases, SuMo detects one common site, which corresponds to the catalytic triad. In the legume lectins family composed of >100 structures that share similar sequences and folds but may have lost their ability to bind a carbohydrate molecule, SuMo discriminates between functional and non-functional lectins with a selectivity of 96%. The time needed for searching a given site in a protein structure is typically 0.1 s on a PIII 800MHz/Linux computer; thus, in further studies, SuMo will be used to screen the PDB.     

Evolutionarily conserved networks of residues mediate allosteric communication in proteins

A fundamental goal in cellular signaling is to understand allosteric communication, the process by which signals originating at one site in a protein propagate reliably to affect distant functional sites. The general principles of protein structure that underlie this process remain unknown. Here, we describe a sequence-based statistical method for quantitatively mapping the global network of amino acid interactions in a protein. Application of this method for three structurally and functionally distinct protein families (G protein-coupled receptors, the chymotrypsin class of serine proteases and hemoglobins) reveals a surprisingly simple architecture for amino acid interactions in each protein family: a small subset of residues forms physically connected networks that link distant functional sites in the tertiary structure. Although small in number, residues comprising the network show excellent correlation with the large body of mechanistic data available for each family. The data suggest that evolutionarily conserved sparse networks of amino acid interactions represent structural motifs for allosteric communication in proteins.     

Phylogenetic Analysis of Proteins Associated in the Four Major Energy Metabolism Systems: Photosynthesis,Aerobic Respiration,Denitrification, and Sulfur Respiration

The four electron transfer energy metabolism systems, photosynthesis, aerobic respiration, denitrification, and sulfur respiration, are thought to be evolutionarily related because of the similarity of electron transfer patterns and the existence of some homologous proteins. How these systems have evolved is elusive. We therefore conducted a comprehensive homology search using PSI-BLAST, and phylogenetic analyses were conducted for the three homologous groups (groups 1–3) based on multiple alignments of domains defined in the Pfam database. There are five electron transfer types important for catalytic reaction in group 1, and many proteins bind molybdenum. Deletions of two domains led to loss of the function of binding molybdenum and ferredoxin, and these deletions seem to be critical for the electron transfer pattern changes in group 1. Two types of electron transfer were found in group 2, and all its member proteins bind siroheme and ferredoxin. Insertion of the pyridine nucleotide disulfide oxidoreductase domain seemed to be the critical point for the electron transfer pattern change in this group. The proteins belonging to group 3 are all flavin enzymes, and they bind flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). Types of electron transfer in this group are divergent, but there are two common characteristics. NAD(P)H works as an electron donor or acceptor, and FAD or FMN transfers electrons from/to NAD(P)H. Electron transfer functions might be added to these common characteristics by the addition of functional domains through the evolution of group 3 proteins. Based on the phylogenetic analyses in this study and previous studies, we inferred the phylogeny of the energy metabolism systems as follows: photosynthesis (and possibly aerobic respiration) and the sulfur/nitrogen assimilation system first diverged, then the sulfur/nitrogen dissimilation system was produced from the latter system.     

Intrinsic disorder in S100 proteins

Although the members of the largest subfamily of the EF-hand proteins, S100 proteins, are evolutionarily young, their functional diversity is extremely broad, partly due to their ability to adapt to various targets. This feature is a hallmark of intrinsically disordered proteins (IDPs), but none of the S100 proteins are recognized as IDPs. S100 are predicted to be enriched in intrinsic disorder, with 62% of them being predicted to be disordered by at least one of the predictors: 31% are recognized as 'molten globules' and 15% are shown to be in extended disordered form. The disorder level of predicted disordered S100 regions is conserved compared to that of more structured regions. The central disordered stretch corresponds to the major part of pseudo EF-hand loop, helix II, hinge region, and an initial part of helix III. It contains about half of known sites of enzymatic post-translational modifications (PTMs), confirming that this region can be flexible in vivo. Most of the internal residues missing in tertiary structures belong to the hinge. Both hinge and pseudo EF-hand loop correspond to the local maxima of the PONDR? VSL2 score and are shown to be evolutionary hotspots, leading to gain of new functional properties. The action of PTMs is shown to be destabilizing, in contrast with the effect of metal-binding or S100 dimerization. Formation of the S100 heterodimers relies on the interplay between the structural rigidity of one of the S100 monomers and the flexibility of another monomer. The ordered regions dominate in the S100 homodimerization sites. Target-binding sites generally consist of distant regions, drastically differing in their disorder level. The disordered region comprising most of the hinge and the N-terminal half of helix III is virtually not involved into dimerization, being intended solely for target recognition. The structural flexibility of this region is essential for recognition of diverse target proteins. At least 86% of multiple interactions of S100 proteins with binding partners are attributed to the S100 proteins predicted to be disordered. Overall, the intrinsic disorder is inherent to many S100 proteins and is vital for activity and functional diversity of the family.     

Domain stability and metal-induced folding of calcium- and integrin-binding protein 1

It is widely accepted that a pair of EF-hands is the functional unit of typical four EF-hand proteins such as calmodulin or troponin C. In this work we investigate the structure and stability of the four EF-hand domains in the related protein calcium- and integrin-binding protein 1 (CIB1) in the presence and absence of Mg2+ or Ca2+, to determine if similar EF-hand interactions occur. The backbone structure and flexibility of CIB1 were first studied by NMR spectroscopy, and these studies were complimented with steady-state fluorescence spectroscopy and chemical denaturation experiments using mutant CIB1 proteins having single Trp reporter groups in each of the four EF-hand domains EF-I (F34W), EF-II (F91W), EF-III (L128W), and EF-IV (F173W). We find that Mg2+-CIB1 adopts a well-folded structure similar to Ca2+-CIB1, except for some conformational heterogeneity in the C-terminal EF-IV domain. The structure of apo-CIB1 is significantly more dynamic, especially within EF-II, EF-III, and a partially unfolded EF-IV region, but the N-terminal EF-I region of apo-CIB1 has a well-ordered and more stable structure. The data reveal significant communication between the N- and C-lobes of CIB1, and show that transient intermediate conformations are formed along the unfolding pathway for each form of the protein. Collectively the data demonstrate that the communication between the paired EF-hand domains as well as between the N- and C-lobes of CIB1 is distinct from the ancestral proteins calmodulin and troponin C, which might be important for the unique function of CIB1 in numerous biological processes.     

Structural and functional similarity between fish antifreeze proteins and calcium-dependent lectins.

A cDNA for a type II antifreeze protein was isolated from liver of smelt (Osmerus mordax). The predicted protein sequence is homologous to that from sea raven (Hemitripterus americanus) and both show homology to a family of calcium-dependent lectins. Smelt and sea raven belong to taxonomic orders believed to have diverged prior to Cenozoic glaciation. Thus, type II antifreeze proteins appear to have evolved independently in these fish species from pre-existing calcium-dependent lectins. Sequence alignment of the antifreezes and the lectins suggest that these proteins adopt a similar fold, that the sea raven antifreeze has lost its Ca2+ binding sites, and the smelt antifreeze has retained one site. Experiments show that smelt antifreeze protein activity is responsive to Ca2+ but that of sea raven antifreeze protein is not. These results suggest that the type II fish antifreeze proteins and calcium-dependent lectins share a common ancestry, related folding structures, and functional similarity.     

Structure-function relationship of monocot mannose-binding lectins.

The monocot mannose-binding lectins are an extended superfamily of structurally and evolutionarily related proteins, which until now have been isolated from species of the Amaryllidaceae, Alliaceae, Araceae, Orchidaceae, and Liliaceae. To explain the obvious differences in biological activities, the structure-function relationships of the monocot mannose-binding lectins were studied by a combination of glycan-binding studies and molecular modeling using the deduced amino acid sequences of the currently known lectins. Molecular modeling indicated that the number of active mannose-binding sites per monomer varies between three and zero. Since the number of binding sites is fairly well correlated with the binding activity measured by surface plasmon resonance, and is also in good agreement with the results of previous studies of the biological activities of the mannose-binding lectins, molecular modeling is of great value for predicting which lectins are best suited for a particular application.     

Comparing graph representations of protein structure for mining family-specific residue-based packing motifs.

We find recurring amino-acid residue packing patterns, or spatial motifs, that are characteristic of protein structural families, by applying a novel frequent subgraph mining algorithm to graph representations of protein three-dimensional structure. Graph nodes represent amino acids, and edges are chosen in one of three ways: first, using a threshold for contact distance between residues; second, using Delaunay tessellation; and third, using the recently developed almost-Delaunay edges. For a set of graphs representing a protein family from the Structural Classification of Proteins (SCOP) database, subgraph mining typically identifies several hundred common subgraphs corresponding to spatial motifs that are frequently found in proteins in the family but rarely found outside of it. We find that some of the large motifs map onto known functional regions in two protein families explored in this study, i.e., serine proteases and kinases. We find that graphs based on almost-Delaunay edges significantly reduce the number of edges in the graph representation and hence present computational advantage, yet the patterns extracted from such graphs have a biological interpretation approximately equivalent to that of those extracted from distance based graphs.     

Identification of substrates for transglutaminase in Physarum polycephalum, an acellular slime mold, upon cellular mechanical damage

Transglutaminases are Ca(2+)-dependent enzymes that post-translationally modify proteins by crosslinking or polyamination at specific polypeptide-bound glutamine residues. Physarum polycephalum, an acellular slime mold, is the evolutionarily lowest organism expressing a transglutimase whose primary structure is similar to that of mammalian transglutimases. We observed transglutimase reaction products at injured sites in Physarum macroplasmodia upon mechanical damage. With use of a biotin-labeled primary amine, three major proteins constituting possible transglutimase substrates were affinity-purified from the damaged slime mold. The purified proteins were Physarum actin, a 40 kDa Ca(2+)-binding protein with four EF-hand motifs (CBP40), and a novel 33 kDa protein highly homologous to the eukaryotic adenine nucleotide translocator, which is expressed in mitochondria. Immunochemical analysis of extracts from the damaged macroplasmodia indicated that CBP40 is partly dimerized, whereas the other proteins migrated as monomers on SDS/PAGE. Of the three proteins, CBP40 accumulated most significantly around injured areas, as observed by immunofluoresence. These results suggested that transglutimase reactions function in the response to mechanical injury.     

Structural alignment of ferredoxin and flavodoxin based on electrostatic potentials: implications for their interactions with photosystem I and ferredoxin-NADP reductase

The two proteins ferredoxin and flavodoxin can replace each other in the photosynthetic electron transfer chain of cyanobacteria and algae. However, structure, size, and composition of ferredoxin and flavodoxin are completely different. Ferredoxin is a small iron-sulfur protein (approximately 100 amino acids), whereas flavodoxin is a flavin-containing protein (approximately 170 amino acids). The crystal structure of both proteins from the cyanobacteria Anabeana PCC 7120 is known. We used these two protein structures to investigate the structural basis of their functional equivalence. We apply the Hodgkin index to quantify the similarity of their electrostatic potentials. The technique has been applied successfully in indirect drug design for the alignment of small molecule and bioisosterism elucidation. It requires no predefined atom-atom correspondences. As is known from experiments, electrostatic interactions are most important for the association of ferredoxin and flavodoxin with their reaction partners photosystem I and ferredoxin-NADP reductase. Therefore, use of electrostatic potentials for the structural alignment is well justified. Our extensive search of the alignment space reveals two alignments with a high degree of similarity in the electrostatic potential. In both alignments, ferredoxin overlaps completely with flavodoxin. The active sites of ferredoxin and flavodoxin rather than their centers of mass coincide in both alignments. This is in agreement with electron microscopy investigations on photosystem I cross-linked to ferredoxin or flavodoxin. We identify residues that may have the same function in both proteins and relate our results to previous experimental data.     

Biological insights from topology independent comparison of protein 3D structures

Comparing and classifying the three-dimensional (3D) structures of proteins is of crucial importance to molecular biology, from helping to determine the function of a protein to determining its evolutionary relationships. Traditionally, 3D structures are classified into groups of families that closely resemble the grouping according to their primary sequence. However, significant structural similarities exist at multiple levels between proteins that belong to these different structural families. In this study, we propose a new algorithm, CLICK, to capture such similarities. The method optimally superimposes a pair of protein structures independent of topology. Amino acid residues are represented by the Cartesian coordinates of a representative point (usually the C(α) atom), side chain solvent accessibility, and secondary structure. Structural comparison is effected by matching cliques of points. CLICK was extensively benchmarked for alignment accuracy on four different sets: (i) 9537 pair-wise alignments between two structures with the same topology; (ii) 64 alignments from set (i) that were considered to constitute difficult alignment cases; (iii) 199 pair-wise alignments between proteins with similar structure but different topology; and (iv) 1275 pair-wise alignments of RNA structures. The accuracy of CLICK alignments was measured by the average structure overlap score and compared with other alignment methods, including HOMSTRAD, MUSTANG, Geometric Hashing, SALIGN, DALI, GANGSTA(+), FATCAT, ARTS and SARA. On average, CLICK produces pair-wise alignments that are either comparable or statistically significantly more accurate than all of these other methods. We have used CLICK to uncover relationships between (previously) unrelated proteins. These new biological insights include: (i) detecting hinge regions in proteins where domain or sub-domains show flexibility; (ii) discovering similar small molecule binding sites from proteins of different folds and (iii) discovering topological variants of known structural/sequence motifs. Our method can generally be applied to compare any pair of molecular structures represented in Cartesian coordinates as exemplified by the RNA structure superimposition benchmark.     

Inferring functional relationships of proteins from local sequence and spatial surface patterns

We describe a novel approach for inferring functional relationship of proteins by detecting sequence and spatial patterns of protein surfaces. Well-formed concave surface regions in the form of pockets and voids are examined to identify similarity relationship that might be directly related to protein function. We first exhaustively identify and measure analytically all 910,379 surface pockets and interior voids on 12,177 protein structures from the Protein Data Bank. The similarity of patterns of residues forming pockets and voids are then assessed in sequence, in spatial arrangement, and in orientational arrangement. Statistical significance in the form of E and p-values is then estimated for each of the three types of similarity measurements. Our method is fully automated without human intervention and can be used without input of query patterns. It does not assume any prior knowledge of functional residues of a protein, and can detect similarity based on surface patterns small and large. It also tolerates, to some extent, conformational flexibility of functional sites. We show with examples that this method can detect functional relationship with specificity for members of the same protein family and superfamily, as well as remotely related functional surfaces from proteins of different fold structures. We envision that this method can be used for discovering novel functional relationship of protein surfaces, for functional annotation of protein structures with unknown biological roles, and for further inquiries on evolutionary origins of structural elements important for protein function.