Crystal structure of the Drosophila peptidoglycan recognition protein (PGRP)-SA at 1.56 A resolution

Peptidoglycan recognition proteins (PGRPs) form a recently discovered protein family, which is conserved from insect to mammals and is implicated in the innate immune system by interacting with/or degrading microbial peptidoglycans (PGNs). Drosophila PGRP-SA is a member of this family of pattern recognition receptors and is involved in insect Toll activation. We report here the crystal structure of PGRP-SA at 1.56 A resolution, which represents the first example of a "recognition" PGRP. Comparison with the catalytic Drosophila PGRP-LB reveals an overall structure conservation with an L-shaped hydrophilic groove that is likely the PGN carbohydrate core binding site, but further suggests some possible functional homology between recognition and catalytic PGRPs. Consistent with sequence analysis, PGRP-SA does not contain the canonical zinc-binding residues found in catalytic PGRPs. However, substitution of the zinc-binding cysteine residue by serine, along with an altered coordinating histidine residue, assembles a constellation of residues that resembles a modified catalytic triad. The serine/histidine juxtaposition to a threonine residue and a carbonyl oxygen atom, along with conservation of the catalytic water molecule found in PGRP-LB, tantalizingly suggests some hydrolytic function for this member of receptor PGRPs.     

Evaluation of features for catalytic residue prediction in novel folds

Structural genomics projects are determining the three-dimensional structure of proteins without full characterization of their function. A critical part of the annotation process involves appropriate knowledge representation and prediction of functionally important residue environments. We have developed a method to extract features from sequence, sequence alignments, three-dimensional structure, and structural environment conservation, and used support vector machines to annotate homologous and nonhomologous residue positions based on a specific training set of residue functions. In order to evaluate this pipeline for automated protein annotation, we applied it to the challenging problem of prediction of catalytic residues in enzymes. We also ranked the features based on their ability to discriminate catalytic from noncatalytic residues. When applying our method to a well-annotated set of protein structures, we found that top-ranked features were a measure of sequence conservation, a measure of structural conservation, a degree of uniqueness of a residue's structural environment, solvent accessibility, and residue hydrophobicity. We also found that features based on structural conservation were complementary to those based on sequence conservation and that they were capable of increasing predictor performance. Using a family nonredundant version of the ASTRAL 40 v1.65 data set, we estimated that the true catalytic residues were correctly predicted in 57.0% of the cases, with a precision of 18.5%. When testing on proteins containing novel folds not used in training, the best features were highly correlated with the training on families, thus validating the approach to nonhomologous catalytic residue prediction in general. We then applied the method to 2781 coordinate files from the structural genomics target pipeline and identified both highly ranked and highly clustered groups of predicted catalytic residues.     

Altered dynamics upon oligomerization corresponds to key functional sites

It is known that over half of the proteins encoded by most organisms function as oligomeric complexes. Oligomerization confers structural stability and dynamics changes in proteins. We investigate the effects of oligomerization on protein dynamics and its functional significance for a set of 145 multimeric proteins. Using coarse‐grained elastic network models, we inspect the changes in residue fluctuations upon oligomerization and then compare with residue conservation scores to identify the functional significance of these changes. Our study reveals conservation of about ½ of the fluctuations, with ¼ of the residues increasing in their mobilities and ¼ having reduced fluctuations. The residues with dampened fluctuations are evolutionarily more conserved and can serve as orthosteric binding sites, indicating their importance. We also use triosephosphate isomerase as a test case to understand why certain enzymes function only in their oligomeric forms despite the monomer including all required catalytic residues. To this end, we compare the residue communities (groups of residues which are highly correlated in their fluctuations) in the monomeric and dimeric forms of the enzyme. We observe significant changes to the dynamical community architecture of the catalytic core of this enzyme. This relates to its functional mechanism and is seen only in the oligomeric form of the protein, answering why proteins are oligomeric structures. Proteins 2017; 85:1422–1434. © 2017 Wiley Periodicals, Inc.     

Designing thermostable proteins: ancestral mutants of 3-isopropylmalate dehydrogenase designed by using a phylogenetic tree

We have recently developed a new method for designing thermostable proteins using phylogenetic trees of enzymes. In this study, we investigated a method for designing proteins with improved stability using 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus as a model enzyme. We designed 12 mutant enzymes, each having an ancestral amino acid residue that was present in the common ancestor of Bacteria and Archaea. At least six of the 12 ancestral mutants tested showed thermal stability higher than that of the original enzyme. The results supported the hyperthermophilic universal ancestor hypothesis. The effect of ancestral residues on IPMDHs of several organisms and on the related enzyme isocitrate dehydrogenase was summarised and analysed. The effect of an ancestral residue on thermostability did not depend on the degree of conservation of the residue at the site, suggesting that the stabilisation of these mutant proteins is not related to sequence conservation but to the antiquity of the introduced residues. The results suggest also that this method could be an efficient way of designing mutant enzymes with higher thermostability based only on the primary structure and a phylogenetic tree.     

Functional diversity of cysteine residues in proteins and unique features of catalytic redox-active cysteines in thiol oxidoreductases

Thiol-dependent redox systems are involved in regulation of diverse biological processes, such as response to stress, signal transduction, and protein folding. The thiol-based redox control is provided by mechanistically similar, but structurally distinct families of enzymes known as thiol oxidoreductases. Many such enzymes have been characterized, but identities and functions of the entire sets of thiol oxidoreductases in organisms are not known. Extreme sequence and structural divergence makes identification of these proteins difficult. Thiol oxidoreductases contain a redox-active cysteine residue, or its functional analog selenocysteine, in their active sites. Here, we describe computational methods for in silico prediction of thiol oxidoreductases in nucleotide and protein sequence databases and identification of their redox-active cysteines. We discuss different functional categories of cysteine residues, describe methods for discrimination between catalytic and noncatalytic and between redox and non-redox cysteine residues and highlight unique properties of the redox-active cysteines based on evolutionary conservation, secondary and three-dimensional structures, and sporadic replacement of cysteines with catalytically superior selenocysteine residues.     

Accurate Prediction of Protein Catalytic Residues by Side Chain Orientation and Residue Contact Density

Prediction of protein catalytic residues provides useful information for the studies of protein functions. Most of the existing methods combine both structure and sequence information but heavily rely on sequence conservation from multiple sequence alignments. The contribution of structure information is usually less than that of sequence conservation in existing methods. We found a novel structure feature, residue side chain orientation, which is the first structure-based feature that achieves prediction results comparable to that of evolutionary sequence conservation. We developed a structure-based method, Enzyme Catalytic residue SIde-chain Arrangement (EXIA), which is based on residue side chain orientations and backbone flexibility of protein structure. The prediction that uses EXIA outperforms existing structure-based features. The prediction quality of combing EXIA and sequence conservation exceeds that of the state-of-the-art prediction methods. EXIA is designed to predict catalytic residues from single protein structure without needing sequence or structure alignments. It provides invaluable information when there is no sufficient or reliable homology information for target protein. We found that catalytic residues have very special side chain orientation and designed the EXIA method based on the newly discovered feature. It was also found that EXIA performs well for a dataset of enzymes without any bounded ligand in their crystallographic structures.     

Predicting and annotating catalytic residues: an information theoretic approach.

We introduce a computational method to predict and annotate the catalytic residues of a protein using only its sequence information, so that we describe both the residues' sequence locations (prediction) and their specific biochemical roles in the catalyzed reaction (annotation). While knowing the chemistry of an enzyme's catalytic residues is essential to understanding its function, the challenges of prediction and annotation have remained difficult, especially when only the enzyme's sequence and no homologous structures are available. Our sequence-based approach follows the guiding principle that catalytic residues performing the same biochemical function should have similar chemical environments; it detects specific conservation patterns near in sequence to known catalytic residues and accordingly constrains what combination of amino acids can be present near a predicted catalytic residue. We associate with each catalytic residue a short sequence profile and define a Kullback-Leibler (KL) distance measure between these profiles, which, as we show, effectively captures even subtle biochemical variations. We apply the method to the class of glycohydrolase enzymes. This class includes proteins from 96 families with very different sequences and folds, many of which perform important functions. In a cross-validation test, our approach correctly predicts the location of the enzymes' catalytic residues with a sensitivity of 80% at a specificity of 99.4%, and in a separate cross-validation we also correctly annotate the biochemical role of 80% of the catalytic residues. Our results compare favorably to existing methods. Moreover, our method is more broadly applicable because it relies on sequence and not structure information; it may, furthermore, be used in conjunction with structure-based methods.     

Analysis and prediction of the location of catalytic residues in enzymes

The catalytic residues of an enzyme are defined as the amino acids directly involved in chemical catalysis. They mainly act as a general acid--base, electrophilic or nucleophilic catalyst or they polarize and stabilize the transition state. An analysis of the structural features of 36 catalytic residues in 17 enzymes of known structure and with defined mechanism is reported. Residues that bind metal ions (Zn2+ and Cu2+) are considered separately. The features examined are: residue type, location in secondary structure, separation between the residues, accessibility to solvent, intra-protein electrostatic interactions, mobility as evaluated from crystallographic temperature factors, polarity of the environment and the sequence conservation between homologous enzymes of residues that were sequentially or spatially close to the catalytic residue. In general the environment of catalytic residues is similar to that of polar side chains that have low accessibility to solvent. Two algorithms have been developed to identify probable catalytic residues. Scanning an alignment of homologous enzyme sequences for peaks of sequence conservation identifies 13 out of the 16 catalytic residues with 50 residues overpredicted. When the conservation of the spatially close residues is used instead, a different set of 13 residues are identified with 47 residues overpredicted. A combination of the two algorithms identifies 11 residues with 36 residues overpredicted.     

Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain.

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.     

Sequence conservation provides the best prediction of the role of proline residues in p13suc1

The unique nature of the proline side-chain imposes severe constraints on the polypeptide backbone, and thus it seems likely that it plays a special structural or functional role in the architecture of proteins. We have investigated the role of proline residues in suc1, a member of the cyclin-dependent kinase (cks) family of proteins, whose known function is to bind to and regulate the activity of the major mitotic cdk. The effect on stability of mutation to alanine of all but two of the eight proline residues is correlated with their conservation within the family. The remaining two proline residues are located in the hinge loop between two beta-strands that mediates a domain-swapping process involving exchange of a beta-strand between two monomers to form a dimer pair. Mutation of these proline residues to alanine stabilises the protein. cdk binding is unaffected by these mutations, but dimerisation is altered. We propose, therefore, that the double-proline motif is conserved for the purpose of domain swapping, which suggests that this phenomenon plays a role in the function of cks proteins. Thus, the conservation of the proline residues is a good indicator of their roles in suc1, either in the stabilisation of the native state or in performing functions that are as yet unknown. In addition, the strain resulting from two of the proline residues was relieved successfully by mutation of the preceeding residue to glycine, suggesting a general method for designing more stable proteins.     

Identification of essential residues in apolipoprotein N-acyl transferase, a member of the CN hydrolase family

Apolipoprotein N-acyl transferase (Lnt) is an essential membrane-bound protein involved in lipid modification of all lipoproteins in gram-negative bacteria. Essential residues in Lnt of Escherichia coli were identified by using site-directed mutagenesis and an in vivo complementation assay. Based on sequence conservation and known protein structures, we predict a model for Lnt, which is a member of the CN hydrolase family. Besides the potential catalytic triad E267-K335-C387, four residues that directly affect the modification of Braun's lipoprotein Lpp are absolutely required for Lnt function. Residues Y388 and E389 are part of the hydrophobic pocket that constitutes the active site. Residues W237 and E343 are located on two flexible arms that face away from the active site and are expected to open and close upon the binding and release of phospholipid and/or apolipoprotein. Substitutions causing temperature-dependent effects were located at different positions in the structural model. These mutants were not affected in protein stability. Lnt proteins from other proteobacteria, but not from actinomycetes, were functional in vivo, and the essential residues identified in Lnt of E. coli are conserved in these proteins.     

A novel protein kinase-like domain in a selenoprotein, widespread in the tree of life

Selenoproteins serve important functions in many organisms, usually providing essential oxidoreductase enzymatic activity, often for defense against toxic xenobiotic substances. Most eukaryotic genomes possess a small number of these proteins, usually not more than 20. Selenoproteins belong to various structural classes, often related to oxidoreductase function, yet a few of them are completely uncharacterised.Here, the structural and functional prediction for the uncharacterised selenoprotein O (SELO) is presented. Using bioinformatics tools, we predict that SELO protein adopts a three-dimensional fold similar to protein kinases. Furthermore, we argue that despite the lack of conservation of the "classic" catalytic aspartate residue of the archetypical His-Arg-Asp motif, SELO kinases might have retained catalytic phosphotransferase activity, albeit with an atypical active site. Lastly, the role of the selenocysteine residue is considered and the possibility of an oxidoreductase-regulated kinase function for SELO is discussed.The novel kinase prediction is discussed in the context of functional data on SELO orthologues in model organisms, FMP40 a.k.a.YPL222W (yeast), and ydiU (bacteria). Expression data from bacteria and yeast suggest a role in oxidative stress response. Analysis of genomic neighbourhoods of SELO homologues in the three domains of life points toward a role in regulation of ABC transport, in oxidative stress response, or in basic metabolism regulation. Among bacteria possessing SELO homologues, there is a significant over-representation of aquatic organisms, also of aerobic ones. The selenocysteine residue in SELO proteins occurs only in few members of this protein family, including proteins from Metazoa, and few small eukaryotes (Ostreococcus, stramenopiles). It is also demonstrated that enterobacterial mchC proteins involved in maturation of bactericidal antibiotics, microcins, form a distant subfamily of the SELO proteins.The new protein structural domain, with a putative kinase function assigned, expands the known kinome and deserves experimental determination of its biological role within the cell-signaling network.     

Cysteine function governs its conservation and degeneration and restricts its utilization on protein surfaces

Cysteine (Cys) is an enigmatic amino acid residue. Although one of the least abundant, it often occurs in the functional sites of proteins. Whereas free Cys is a polar amino acid, Cys in proteins is often buried, and its classification on the hydrophobicity scale is ambiguous. We hypothesized that the deviation of Cys residues from the properties of a free amino acid is due to their reactivity and addressed this possibility by examining Cys in large protein structure data sets. Compared to other amino acids, Cys was characterized by the most extreme conservation pattern, with the majority of Cys being either highly conserved or poorly conserved. In addition, clustering of Cys with another Cys residue was associated with high conservation, whereas exposure of Cys on protein surfaces was associated with low conservation. Moreover, although clustered Cys behaved as polar residues, isolated Cys was the most buried residue of all, in disagreement with known chemical properties of Cys. Thus, the anomalous hydrophobic behavior and conservation pattern of Cys can be explained by elimination of isolated Cys from protein surfaces during evolution and by clustering of other Cys residues. These findings indicate that Cys abundance is governed by Cys function in proteins rather than by the sheer chemical-physical properties of free amino acids, and suggest that a high tendency of Cys to be functionally active can considerably limit its abundance on protein surfaces.     

A conservation and rigidity based method for detecting critical protein residues

Background

Certain amino acids in proteins play a critical role in determining their structural stability and function. Examples include flexible regions such as hinges which allow domain motion, and highly conserved residues on functional interfaces which allow interactions with other proteins. Detecting these regions can aid in the analysis and simulation of protein rigidity and conformational changes, and helps characterizing protein binding and docking. We present an analysis of critical residues in proteins using a combination of two complementary techniques. One method performs in-silico mutations and analyzes the protein's rigidity to infer the role of a point substitution to Glycine or Alanine. The other method uses evolutionary conservation to find functional interfaces in proteins.

Results

We applied the two methods to a dataset of proteins, including biomolecules with experimentally known critical residues as determined by the free energy of unfolding. Our results show that the combination of the two methods can detect the vast majority of critical residues in tested proteins.

Conclusions

Our results show that the combination of the two methods has the potential to detect more information than each method separately. Future work will provide a confidence level for the criticalness of a residue to improve the accuracy of our method and eliminate false positives. Once the combined methods are integrated into one scoring function, it can be applied to other domains such as estimating functional interfaces.

     

A Structure-Based Approach for Detection of Thiol Oxidoreductases and Their Catalytic Redox-Active Cysteine Residues

Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, targeting proteins to the membranes, and various catalytic functions. However, the structural determinants for various Cys functions are not clear. Thiol oxidoreductases, which are enzymes containing catalytic redox-active Cys residues, have been extensively studied, but even for these proteins there is little understanding of what distinguishes their catalytic redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can recognize these enzymes by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Cys and (ii) calculating accessibility, active site location, and reactivity of Cys. For proteins with known or modeled structures, this method can identify proteins with catalytic Cys residues and distinguish thiol oxidoreductases from the enzymes containing other catalytic Cys types. Furthermore, by applying this procedure to Saccharomyces cerevisiae proteins containing conserved Cys, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of catalytic redox-active Cys and should further help to recognize thiol oxidoreductases in protein sequence and structure databases.     

Analysis of homodimeric protein interfaces by graph-spectral methods

The quaternary structures impart structural and functional credibility to proteins. In a multi-subunit protein, it is important to understand the factors that drive the association or dissociation of the subunits. It is a well known fact that both hydrophobic and charged interactions contribute to the stability of the protein interface. The interface residues are also known to be highly conserved. Though they are buried in the oligomer, these residues are either exposed or partially exposed in the monomer. It is felt that a systematic and objective method of identifying interface clusters and their analysis can significantly contribute to the identification of a residue or a collection of residues important for oligomerization. Recently, we have applied the techniques of graph-spectral methods to a variety of problems related to protein structure and folding. A major advantage of this methodology is that the problem is viewed from a global protein topology point of view rather than localized regions of the protein structure. In the present investigation, we have applied the methods of graph-spectral analysis to identify side chain clusters at the interface and the centers of these clusters in a set of homodimeric proteins. These clusters are analyzed in terms of properties such as amino acid composition, accessibility to solvent and conservation of residues. Interesting results such as participation of charged and aromatic residues like arginine, glutamic acid, histidine, phenylalanine and tyrosine, consistent with earlier investigations, have emerged from these analyses. Important additional information is that the residues involved are a part of a cluster(s) and that they are sequentially distant residues which have come closer to each other in the three-dimensional structure of the protein. These residues can easily be detected using our graph-spectral algorithm. This method has also been used to identify important residues ('hot spots') in dimerization and also to detect dimerization sites on the monomer. The residues predicted using the present algorithm have correlated well with the experiments indicating the efficacy of this method in predicting residues involved in dimer stability.     

Isolation, characterization, sequencing and crystal structure of charybdin, a type 1 ribosome-inactivating protein from Charybdis maritima agg

A novel, type 1 ribosome-inactivating protein designated charybdin was isolated from bulbs of Charybdis maritima agg. The protein, consisting of a single polypeptide chain with a molecular mass of 29 kDa, inhibited translation in rabbit reticulocytes with an IC50 of 27.2 nm. Plant genomic DNA extracted from the bulb was amplified by PCR between primers based on the N-terminal and C-terminal sequence of the protein from dissolved crystals. The complete mature protein sequence was derived by partial DNA sequencing and terminal protein sequencing, and was confirmed by high-resolution crystal structure analysis. The protein contains Val at position 79 instead of the conserved Tyr residue of the ribosome-inactivating proteins known to date. To our knowledge, this is the first observation of a natural substitution of a catalytic residue at the active site of a natural ribosome-inactivating protein. This substitution in the active site may be responsible for the relatively low in vitro translation inhibitory effect compared with other ribosome-inactivating proteins. Single crystals were grown in the cold room from PEG6000 solutions. Diffraction data collected to 1.6 A resolution were used to determine the protein structure by the molecular replacement method. The fold of the protein comprises two structural domains: an alpha + beta N-terminal domain (residues 4-190) and a mainly alpha-helical C-terminal domain (residues 191-257). The active site is located in the interface between the two domains and comprises residues Val79, Tyr117, Glu167 and Arg170.     

Charged residues at protein interaction interfaces: unexpected conservation and orchestrated divergence

Protein-protein interactions play an essential role in the functioning of cell. The importance of charged residues and their diverse role in protein-protein interactions have been well studied using experimental and computational methods. Often, charged residues located in protein interaction interfaces are conserved across the families of homologous proteins and protein complexes. However, on a large scale, it has been recently shown that charged residues are significantly less conserved than other residue types in protein interaction interfaces. The goal of this work is to understand the role of charged residues in the protein interaction interfaces through their conservation patterns. Here, we propose a simple approach where the structural conservation of the charged residue pairs is analyzed among the pairs of homologous binary complexes. Specifically, we determine a large set of homologous interactions using an interaction interface similarity measure and catalog the basic types of conservation patterns among the charged residue pairs. We find an unexpected conservation pattern, which we call the correlated reappearance, occurring among the pairs of homologous interfaces more frequently than the fully conserved pairs of charged residues. Furthermore, the analysis of the conservation patterns across different superkingdoms as well as structural classes of proteins has revealed that the correlated reappearance of charged residues is by far the most prevalent conservation pattern, often occurring more frequently than the unconserved charged residues. We discuss a possible role that the new conservation pattern may play in the long-range electrostatic steering effect.     

Crystal structure of cold-active protein-tyrosine phosphatase from a psychrophile, Shewanella sp

The cold-active protein-tyrosine phosphatase (CAPTPase) of a psychrophile, Shewanella sp., shows high catalytic activity below 20 degrees C. The catalytic residue of CAPTPase is histidine, as opposed to the cysteine of known protein-tyrosine phosphatases (PTPases), and the enzyme protein has three amino acid sequences, Asp-Xaa-His, Gly-Asp-Xaa-Xaa-Asp-Arg and Gly-Asn-His-Glu, that are observed in many protein-serine/threonine phosphatases (PS/TPases). We have determined the crystal structures of CAPTPase at 1.82 angstroms and the enzyme bound with a phosphate ion at 1.90 angstroms resolution using X-ray crystallography and the multiple isomorphous replacement method. The final refined models are comprised of 331 amino acid residues, two metal ions, 447 water molecules, and an acetate or phosphate ion in an asymmetric unit. The enzyme protein consists of three beta-sheets, termed Sheet I, Sheet I', and Sheet II, and 14 alpha-helices. The CAPTPase has a different overall structure from known protein-tyrosine phosphatases. The arrangement of two metal ions, a phosphate ion and the adjacent amino acid residues in the catalytic site of CAPTPase is identical to that of PS/TPases. Thus, it was confirmed that the CAPTPase was a novel PTPase with a conformation similar to the catalytic site of PS/TPase. We speculate that the hydrophobic moiety around the catalytic residue of CAPTPase might play an important role in eliciting high activity at low temperature.