Template-based recognition of protein fold within the midnight and twilight zones of protein sequence similarity

Most homologous pairs of proteins have no significant sequence similarity to each other and are not identified by direct sequence comparison or profile-based strategies. However, multiple sequence alignments of low similarity homologues typically reveal a limited number of positions that are well conserved despite diversity of function. It may be inferred that conservation at most of these positions is the result of the importance of the contribution of these amino acids to the folding and stability of the protein. As such, these amino acids and their relative positions may define a structural signature. We demonstrate that extraction of this fold template provides the basis for the sequence database to be searched for patterns consistent with the fold, enabling identification of homologs that are not recognized by global sequence analysis. The fold template method was developed to address the need for a tool that could comprehensively search the midnight and twilight zones of protein sequence similarity without reliance on global statistical significance. Manual implementations of the fold template method were performed on three folds--immunoglobulin, c-lectin and TIM barrel. Following proof of concept of the template method, an automated version of the approach was developed. This automated fold template method was used to develop fold templates for 10 of the more populated folds in the SCOP database. The fold template method developed three-dimensional structural motifs or signatures that were able to return a diverse collection of proteins, while maintaining a low false positive rate. Although the results of the manual fold template method were more comprehensive than the automated fold template method, the diversity of the results from the automated fold template method surpassed those of current methods that rely on statistical significance to infer evolutionary relationships among divergent proteins.     

Consistency analysis of similarity between multiple alignments: prediction of protein function and fold structure from analysis of local sequence motifs

A new method to analyze the similarity between multiply aligned protein motifs (blocks) was developed. It identifies sets of consistently aligned blocks. These are found to be protein regions of similar function and structure that appear in different contexts. For example, the Rossmann fold ligand-binding region is found similar to TIM barrel and methylase regions, various protein families are predicted to have a TIM-barrel fold and the structural relation between the ClpP protease and crotonase folds is identified from their sequence. Besides identifying local structure features, sequence similarity across short sequence-regions (less than 20 amino acid regions) also predicts structure similarity of whole domains (folds) a few hundred amino acid residues long. Most of these relations could not be identified by other advanced sequence-to-sequence or sequence-to-multiple alignments comparisons. We describe the method (termed CYRCA), present examples of our findings, and discuss their implications.     

Homology among (betaalpha)(8) barrels: implications for the evolution of metabolic pathways

We provide statistically reliable sequence evidence indicating that at least 12 of 23 SCOP (betaalpha)(8) (TIM) barrel superfamilies share a common origin. This includes all but one of the known and predicted TIM barrels found in central metabolism. The statistical evidence is complemented by an examination of the details of protein structure, with certain structural locations favouring catalytic residues even though the nature of their molecular function may change. The combined analysis of sequence, structure and function also enables us to propose a phylogeny of TIM barrels. Based on these data, we are able to examine differing theories of pathway and enzyme evolution, by mapping known TIM barrel folds to the pathways of central metabolism. The results favour widespread recruitment of enzymes between pathways, rather than a "backwards evolution" model, and support the idea that modern proteins may have arisen from common ancestors that bound key metabolites.     

AbrB-like transcription factors assume a swapped hairpin fold that is evolutionarily related to double-psi beta barrels

AbrB is a key transition-state regulator of Bacillus subtilis. Based on the conservation of a betaalphabeta structural unit, we proposed a beta barrel fold for its DNA binding domain, similar to, but topologically distinct from, double-psi beta barrels. However, the NMR structure revealed a novel fold, the "looped-hinge helix." To understand this discrepancy, we undertook a bioinformatics study of AbrB and its homologs; these form a large superfamily, which includes SpoVT, PrlF, MraZ, addiction module antidotes (PemI, MazE), plasmid maintenance proteins (VagC, VapB), and archaeal PhoU homologs. MazE and MraZ form swapped-hairpin beta barrels. We therefore reexamined the fold of AbrB by NMR spectroscopy and found that it also forms a swapped-hairpin barrel. The conservation of the core betaalphabeta element supports a common evolutionary origin for swapped-hairpin and double-psi barrels, which we group into a higher-order class, the cradle-loop barrels, based on the peculiar shape of their ligand binding site.     

Crystal structure of a methyltetrahydrofolate- and corrinoid-dependent methyltransferase

BACKGROUND: Methyltetrahydrofolate, corrinoid iron-sulfur protein methyltransferase (MeTr), catalyzes a key step in the Wood-Ljungdahl pathway of carbon dioxide fixation. It transfers the N5-methyl group from methyltetrahydrofolate (CH3-H4folate) to a cob(I)amide center in another protein, the corrinoid iron-sulfur protein. MeTr is a member of a family of proteins that includes methionine synthase and methanogenic enzymes that activate the methyl group of methyltetra-hydromethano(or -sarcino)pterin. We report the first structure of a protein in this family. RESULTS: We determined the crystal structure of MeTr from Clostridium thermoaceticum at 2.2 A resolution using multiwavelength anomalous diffraction methods. The overall architecture presents a new functional class of the versatile triose phosphate isomerase (TIM) barrel fold. The MeTr tertiary structure is surprisingly similar to the crystal structures of dihydropteroate synthetases despite sharing less than 20% sequence identity. This homology permitted the methyl-H4folate binding site to be modeled. The model suggests extensive conservation of the pterin ring binding residues in the polar active sites of the methyltransferases and dihydropteroate synthetases. The most significant structural difference between these enzymes is in a loop structure above the active site. It is quite open in MeTr, where it can be modeled as the cobalamin binding site. CONCLUSIONS: The MeTr structure consists of a TIM barrel that embeds methyl-H4folate and cobamide. All related methyltransferases are predicted to fold into a similar TIM barrel pattern and have a similar pterin and cobamide binding site. The observed structure is consistent with either a 'front' (N5) or 'back' (C8a) side protonation of CH3-H4folate, a key step that enhances the electrophilic character of the methyl group, activating it for nucleophilic attack by Co(I).     

Toward the detection and validation of repeats in protein structure

We present a method called DAVROS to detect, localize, and validate repeating motifs in protein structure allowing for insertions and deletions. DAVROS uses the score matrix from a structural alignment program (SAP) to search for repeating motifs using an algorithm based on concepts from signal processing and the statistical properties of the alignments. The method was tested against a nonredundant Protein Data Bank, and each chain was assigned a score. For the top 50 chains ranked by score, 70% contain repeating motifs detected without error. These represent 14 types of fold covering alpha, beta, and alphabeta protein classes. A second data set comprising protein chains in different sequence families for triosephosphate isomerase (TIM) barrel, leucine-rich repeat (LRR), trefoil, and alpha-alpha barrel folds was used to assess the ability of DAVROS to detect all motifs within a specific fold. For the second test set, the percentage of motifs detected was highest for the LRR chains (88.7%) and least for the TIM barrels (60%). This variability results from the regularity of the LRR motif compared to the alphabeta units of the TIM barrel, which generally have many more indels. These reduce the strength of the repeat signal in the SAP matrix, making repeat detection more difficult.     

Molecular modeling of 2-nitropropane dioxygenase domain of Mycobacterium tuberculosis H37Rv and docking of herbal ligands

The 3D structure of enoyl reductase (ER) domain generated by the SWISS MODEL server contains the 2-nitropropane dioxygenase (2NPD) structure displaying the TIM barrel fold. Though TIM barrel fold is made up of both main and inserted domains, in our study, we could only predict the structure of the main domain, which had central barrel of eight beta-strands surrounded by eight alpha-helices. Superimposition of the 2NPD region of ER domain of Mycobacterium tuberculosis H37Rv on to the corresponding region of 2UVA_G revealed a good structural alignment between the two, suggesting this template to be a good structural homologue. Among various herbal ligands that were screened as inhibitors, daucosterol was found to bind in closest proximity to the flavin mono nucleotide (FMN) binding site with the lowest docking energy.     

Dipole moment in TIM alpha/beta fold proteins

TIM proteins of alpha/beta barrel fold from alpha/beta class as given in SCOP database were taken for dipole moment analysis. In all, 32 structures were analyzed for their dipole moment contributions. Representative structures from 20 super families in the alpha/beta fold, with different enzyme functions and 12 protein domains of TIM family in TIM super family were considered. The active sites of these proteins are located on the C-terminal side of the beta-strands. The molecules of same alpha/beta fold, but differing in their functionality also showed a common electrostatic field pattern along the barrel axis and had the dipole moment along the barrel axis and towards C-terminal end of the beta-strands. However, it is observed from our calculations that the dipole moment direction is possibly a consequence of the structural fold, with distribution of charges playing a modulatory role, and does not contribute to the location of active site. We show here that apart from the commonly held view as proposed by Hol et al [Hol W G L, van Duijnen PT and Berendsen H J C (1978) Nature (London), 273, 443-446] of the role of the alpha helical dipole moment, the beta-sheets in the barrel can also have a considerable dipole moment contribution. Taken together with our dipole moment analysis on integral membrane proteins [Vasanthi G and Krishnaswamy S (2002) Indian J Biochem Biophys 39, 93-100], this suggests the need to examine the role of dipole moment in the case of especially beta sheets forming barrels.     

The N-terminal β-sheet of the hyperthermophilic endoglucanase from Pyrococcus horikoshii is critical for thermostability

The β-1,4-endoglucanase (EC 3.2.1.4) from the hyperthermophilic archaeon Pyrococcus horikoshii (EGPh) has strong hydrolyzing activity toward crystalline cellulose. When EGPh is used in combination with β-glucosidase (EC 3.2.1.21), cellulose is completely hydrolyzed to glucose at high temperature, suggesting great potential for EGPh in bioethanol industrial applications. The crystal structure of EGPh shows a triosephosphate isomerase (TIM) (β/α)(8)-barrel fold with an N-terminal antiparallel β-sheet at the opposite side of the active site and a very short C-terminal sequence outside of the barrel structure. We describe here the function of the peripheral sequences outside of the TIM barrel core structure. Sequential deletions were performed from both N and C termini. The activity, thermostability, and pH stability of the expressed mutants were assessed and compared to the wild-type EGPh enzyme. Our results demonstrate that the TIM barrel core is essential for enzyme activity and that the N-terminal β-sheet is critical for enzyme thermostability. Bioinformatics analyses identified potential key residues which may contribute to enzyme hyperthermostability.     

Identification of conserved residue patterns in small beta-barrel proteins

Our abilities to predict three-dimensional conformation of a polypeptide, given its amino acid sequence, remain limited despite advances in structure analysis. Analysis of structures and sequences of protein families with similar secondary structural elements, but varying topologies, might help in addressing this problem. We have studied the small beta-barrel class of proteins characterized by four strands (n = 4) and a shear number of 8 (S = 8) to understand the principles of barrel formation. Multiple alignments of the various protein sequences were generated for the analysis. Positional entropy, as a measure of residue conservation, indicated conservation of non-polar residues at the core positions. The presence of a type II beta-turn among the various barrel proteins considered was another strikingly invariant feature. A conserved glycyl-aspartyl dipeptide at the beta-turn appeared to be important in guiding the protein sequence into the barrel fold. Molecular dynamics simulations of the type II beta-turn peptide suggested that aspartate is a key residue in the folding of the protein sequence into the barrel. Our study suggests that the conserved type II beta-turn and the non-polar residues in the barrel core are crucial for the folding of the protein's primary sequence into the beta-barrel conformation.     

Partial NMR assignments and secondary structure mapping of the isolated alpha subunit of Escherichia coli tryptophan synthase,a 29-kD TIM barrel protein

The alpha subunit of tryptophan synthase (alphaTS) from S. typhimurium belongs to the triosephosphate isomerase (TIM) or the (beta/alpha)(8) barrel fold, one of the most common structures in biology. To test the conservation of the global fold in the isolated Escherichia coli homolog, we have obtained a majority of the backbone assignments for the 29-kD alphaTS by using standard heteronuclear multidimensional NMR methods on uniformly (15)N- and (15)N/(13)C-labeled protein and on protein selectively (15)N-labeled at key hydrophobic residues. The secondary structure mapped by chemical shift index, nuclear Overhauser enhancements (NOEs), and hydrogen-deuterium (H-D) exchange, and several abnormal chemical shifts are consistent with the conservation of the global TIM barrel fold of the isolated E. coli alphaTS. Because most of the amide protons that are slow to exchange with solvent correspond to the beta-sheet residues, the beta-barrel is likely to play an important role in stabilizing the previously detected folding intermediates for E. coli alphaTS. A similar combination of uniform and selective labeling can be extended to other TIM barrel proteins to obtain insight into the role of the motif in stabilizing what appear to be common partially folded forms.     

New structural insights into lectin-type proteins of the immune system.

New structural data have emerged for the ligand-binding sites of C-type lectin domains and C-type lectin-like domains of receptors of the immune system. These include binding sites for oligosaccharide or polypeptide ligands, or both oligosaccharide and polypeptide ligands. The structural basis for the binding of a lectin domain of the beta-trefoil family to different sulfooligosaccharide sequences has been revealed. Lectin activity has been documented for a beta/alpha TIM barrel fold that does not have the chitinase activity of the prototype enzyme with this fold.     

Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol

In Neisseria meningitidis and related bacterial pathogens, sialic acids play critical roles in mammalian cell immunity evasion and are synthesized by a conserved enzymatic pathway that includes sialic acid synthase (NeuB, SiaC, or SynC). NeuB catalyzes the condensation of phosphoenolpyruvate (PEP) and N-acetylmannosamine, directly forming N-acetylneuraminic acid (or sialic acid). In this paper we report the development of a coupled assay to monitor NeuB reaction kinetics and an 18O-labeling study that demonstrates the synthase operates via a C-O bond cleavage mechanism. We also report the first structure of a sialic acid synthase, that of NeuB, revealing a unique domain-swapped homodimer architecture consisting of a (beta/alpha)8 barrel (TIM barrel)-type fold at the N-terminal end and a domain with high sequence identity and structural similarity to the ice binding type III antifreeze proteins at the C-terminal end of the enzyme. We have determined the structures of NeuB in the malate-bound form and with bound PEP and the substrate analog N-acetylmannosaminitol to 1.9 and 2.2 A resolution, respectively. Typical of other TIM barrel proteins, the active site of NeuB is located in a cavity at the C-terminal end of the barrel; however, the positioning of the swapped antifreeze-like domain from the adjacent monomer provides key residues for hydrogen bonding with substrates in the active site of NeuB, a structural feature that leads to distinct modes of substrate binding from other PEP-utilizing enzymes that lack an analogous antifreeze-like domain. Our observation of a direct interaction between a highly ordered manganese and the N-acetylmannosaminitol in the NeuB active site also suggests an essential role for the ion as an electrophilic catalyst that activates the N-acetylmannosamine carbonyl to the addition of PEP.     

Similarity in Shape Dictates Signature Intrinsic Dynamics Despite No Functional Conservation in TIM Barrel Enzymes

The conservation of the intrinsic dynamics of proteins emerges as we attempt to understand the relationship between sequence, structure and functional conservation. We characterise the conservation of such dynamics in a case where the structure is conserved but function differs greatly. The triosephosphate isomerase barrel fold (TBF), renowned for its 8 β-strand-α-helix repeats that close to form a barrel, is one of the most diverse and abundant folds found in known protein structures. Proteins with this fold have diverse enzymatic functions spanning five of six Enzyme Commission classes, and we have picked five different superfamily candidates for our analysis using elastic network models. We find that the overall shape is a large determinant in the similarity of the intrinsic dynamics, regardless of function. In particular, the β-barrel core is highly rigid, while the α-helices that flank the β-strands have greater relative mobility, allowing for the many possibilities for placement of catalytic residues. We find that these elements correlate with each other via the loops that link them, as opposed to being directly correlated. We are also able to analyse the types of motions encoded by the normal mode vectors of the α-helices. We suggest that the global conservation of the intrinsic dynamics in the TBF contributes greatly to its success as an enzymatic scaffold both through evolution and enzyme design.     

De novo protein design. II. Plasticity in sequence space.

It is generally accepted that many different protein sequences have similar folded structures, and that there is a relatively high probability that a new sequence possesses a previously observed fold. An indirect consequence of this is that protein design should define the sequence space accessible to a given structure, rather than providing a single optimized sequence. We have recently developed a new approach for protein sequence design, which optimizes the complete sequence of a protein based on the knowledge of its backbone structure, its amino acid composition and a physical energy function including van der Waals interactions, electrostatics, and environment free energy. The specificity of the designed sequence for its template backbone is imposed by keeping the amino acid composition fixed. Here, we show that our procedure converges in sequence space, albeit not to the native sequence of the protein. We observe that while polar residues are well conserved in our designed sequences, non-polar amino acids at the surface of a protein are often replaced by polar residues. The designed sequences provide a multiple alignment of sequences that all adopt the same three-dimensional fold. This alignment is used to derive a profile matrix for chicken triose phosphate isomerase, TIM. The matrix is found to recognize significantly the native sequence for TIM, as well as closely related sequences. Possible application of this approach to protein fold recognition is discussed.     

Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 A resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name "DRE-TIM metallolyases" for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and can guide subsequent studies directed at definitive experimental elucidation of this enzyme's reaction mechanism.     

Interaction energy based protein structure networks

The three-dimensional structure of a protein is formed and maintained by the noncovalent interactions among the amino-acid residues of the polypeptide chain. These interactions can be represented collectively in the form of a network. So far, such networks have been investigated by considering the connections based on distances between the amino-acid residues. Here we present a method of constructing the structure network based on interaction energies among the amino-acid residues in the protein. We have investigated the properties of such protein energy-based networks (PENs) and have shown correlations to protein structural features such as the clusters of residues involved in stability, formation of secondary and super-secondary structural units. Further we demonstrate that the analysis of PENs in terms of parameters such as hubs and shortest paths can provide a variety of biologically important information, such as the residues crucial for stabilizing the folded units and the paths of communication between distal residues in the protein. Finally, the energy regimes for different levels of stabilization in the protein structure have clearly emerged from the PEN analysis.     

Universally conserved positions in protein folds: reading evolutionary signals about stability, folding kinetics and function.

Here, we provide an analysis of molecular evolution of five of the most populated protein folds: immunoglobulin fold, oligonucleotide-binding fold, Rossman fold, alpha/beta plait, and TIM barrels. In order to distinguish between "historic", functional and structural reasons for amino acid conservations, we consider proteins that acquire the same fold and have no evident sequence homology. For each fold we identify positions that are conserved within each individual family and coincide when non-homologous proteins are structurally superimposed. As a baseline for statistical assessment we use the conservatism expected based on the solvent accessibility. The analysis is based on a new concept of "conservatism-of-conservatism". This approach allows us to identify the structural features that are stabilized in all proteins having a given fold, despite the fact that actual interactions that provide such stabilization may vary from protein to protein. Comparison with experimental data on thermodynamics, folding kinetics and function of the proteins reveals that such universally conserved clusters correspond to either: (i) super-sites (common location of active site in proteins having common tertiary structures but not function) or (ii) folding nuclei whose stability is an important determinant of folding rate, or both (in the case of Rossman fold). The analysis also helps to clarify the relation between folding and function that is apparent for some folds.     

An allosteric pathway explains beneficial fitness in yeast for long‐range mutations in an essential TIM barrel enzyme

Protein evolution proceeds by a complex response of organismal fitness to mutations that can simultaneously affect protein stability, structure, and enzymatic activity. To probe the relationship between genotype and phenotype, we chose a fundamental paradigm for protein evolution, folding, and design, the (βα)₈ TIM barrel fold. Here, we demonstrate the role of long‐range allosteric interactions in the adaptation of an essential hyperthermophilic TIM barrel enzyme to mesophilic conditions in a yeast host. Beneficial fitness effects observed with single and double mutations of the canonical βα‐hairpin clamps and the α‐helical shell distal to the active site revealed an underlying energy network between opposite faces of the cylindrical β‐barrel. We experimentally determined the fitness of multiple mutants in the energetic phase plane, contrasting the energy barrier of the chemical reaction and the folding free energy of the protein. For the system studied, the reaction energy barrier was the primary determinant of organism fitness. Our observations of long‐range epistatic interactions uncovered an allosteric pathway in an ancient and ubiquitous enzyme that may provide a novel way of designing proteins with a desired activity and stability profile.