Amino acid sequence analysis of barley stripe mosaic viral proteins: tentative identification of viral serine proteases

Analysis of amino acid sequences of barley stripe mosaic virus (BSMV) proteins revealed the pentapeptide GDSGG, the sequence unique for catalytic centers of serine chymotrypsin-like proteases, in protein p14 encoded by open reading frame 4 of RNA beta. Computer-assisted comparisons revealed a statistically significant similarity between amino acid sequences of p14 and chymotrypsin-like proteases. The catalytic His and Asp residues tentatively identified in p14 together with the Ser residue of the GDSGG sequence, presumably, constitute the "catalytic triad" characteristic of chymotrypsin-like proteases. Based on these observations and on the presence of a potential N-proximal transmembrane domain in p14, this protein may be suggested to be a serine protease involved in processing of the replicase precursor within a membrane-bound replication complex of BSMV.     

Human rhinovirus 3C protease: a cysteine protease showing the trypsin(ogen)-like fold

Viral-encoded proteases cleave precursor polyprotein(s) leading to maturation of infectious virions. Strikingly, human rhinovirus 3C protease shows the trypsin(ogen)-like serine protease fold based on two topologically equivalent six-stranded β-barrels, but displays residue Cys147 as the active site nucleophile. By contrast, papain, which is representative of most cysteine proteases, does not display the trypsin(ogen)-like fold. Remarkably, in human rhinovirus 3C cysteine protease, the catalytic residues Cys147, His40 and Glu71 are positioned as Ser195, His57 and Asp102, respectively, building up the catalytic triad of serine proteases in the chymotrypsin–trypsin–elastase family. However, as compared to trypsin-like serine proteases and their zymogens, residue His40 and the oxyanion hole of human rhinovirus 3C cysteine protease, both key structural components of the active site, are located closer to the protein core. Human rhinovirus 3C cysteine protease cleaves preferentially GlnGly peptide bonds or, less commonly, the GlnSer, GlnAla, GluSer or GluGly pairs. Finally, human rhinovirus 3C cysteine protease and the 3CD cysteine protease–polymerase covalent complex bind the 5′ non-coding region of rhinovirus genomic RNA, an essential function for replication of the viral genome.     

Molecular markers of serine protease evolution.

The evolutionary history of serine proteases can be accounted for by highly conserved amino acids that form crucial structural and chemical elements of the catalytic apparatus. These residues display non- random dichotomies in either amino acid choice or serine codon usage and serve as discrete markers for tracking changes in the active site environment and supporting structures. These markers categorize serine proteases of the chymotrypsin-like, subtilisin-like and alpha/beta-hydrolase fold clans according to phylogenetic lineages, and indicate the relative ages and order of appearance of those lineages. A common theme among these three unrelated clans of serine proteases is the development or maintenance of a catalytic tetrad, the fourth member of which is a Ser or Cys whose side chain helps stabilize other residues of the standard catalytic triad. A genetic mechanism for mutation of conserved markers, domain duplication followed by gene splitting, is suggested by analysis of evolutionary markers from newly sequenced genes with multiple protease domains.     

Ser(214) is crucial for substrate binding to serine proteases

Highly conserved amino acids that form crucial structural elements of the catalytic apparatus can be used to account for the evolutionary history of serine proteases and the cascades into which they are organized. One such evolutionary marker in chymotrypsin-like proteases is Ser(214), located adjacent to the active site and forming part of the primary specificity pocket. Here we report the mutation of Ser(214) in thrombin to Ala, Thr, Cys, Asp, Glu, and Lys. None of the mutants seriously compromises active site catalytic function as measured by the kinetic parameter k(cat). However, the least conservative mutations result in large increases in K(m) because of lower rates of substrate diffusion into the active site. Therefore, the role of Ser(214) is to promote the productive formation of the enzyme-substrate complex. The S214C mutant is catalytically inactive, which suggests that during evolution the TCN-->AGY codon transitions for Ser(214) occurred through Thr intermediates.     

Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases

Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino‐acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases.     

Unconventional serine proteases: variations on the catalytic Ser/His/Asp triad configuration

Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called "classical" catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of "nonclassical" serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.     

Investigating the role of histidine 157 in the catalytic activity of human cytomegalovirus protease

Herpesvirus proteases belong to a new class of serine proteases and contain a novel Ser-His-His catalytic triad, while classical serine proteases have an acidic residue as the third member. To gain a better understanding of the molecular basis for the functional role of the third-member His residue, we have carried out structural and biochemical investigations of human cytomegalovirus (HCMV) protease that bears mutations of the His157 third member. Kinetic studies showed that all the mutants have reduced catalytic activity. Structural studies revealed that a solvent molecule is hydrogen-bonded to the His63 second member and Ser134 in the H157A mutant, partly rescuing the activity of this mutant. This is confirmed by our kinetic and structural observations on the S134A/H157A double mutant, which showed further reductions in the catalytic activity. The structure of the H157A mutant is also in complex with the PMSF inhibitor. The H157E mutant has the best catalytic activity among the mutants; its structure, however, showed conformational readjustments of the His63 and Ser132 residues. The Ser132-His63 diad of HCMV protease has similar activity as the diads in classical serine proteases, whereas the contribution of the His157 third member to the catalysis is much smaller. Finally, structural comparisons revealed the presence of two conserved structural water molecules at the bottom of the S(1) pocket, suggesting a possible new direction for the design of HCMV protease inhibitors.     

Saturation mutagenesis reveals that GLU54 of norovirus 3C-like protease is not essential for the proteolytic activity

The norovirus 3C-like protease is a member of the chymotrypsin-like serine protease superfamily. Previous characterization of its crystal structure has implicated the Glu54-His30-Cys139 triad in the catalysis. In the present study, the Glu54 residue of the protease was subjected to site-saturation mutagenesis, with the result that nearly half of the mutants retained the significant proteolytic activity. It was suggested that a carboxylate at position 54 was not essential for the activity. The in vitro assays of the proteolysis revealed that most of Glu54 mutants retained relatively high proteolytic activity. When the Glu54 mutation was combined with the Ser mutation of the Cys139 residue, a nucleophile, only the Asp54 and Gln54 mutations showed proteolytic activity comparable to that of the Ser139 single mutant, suggesting that a hydrogen bond between Glu54 and His30 was critical in the Ser139 background. These results suggested that the mechanism of the proteolysis by the wild-type norovirus 3C-like protease was different from that of typical chymotrypsin-like serine proteases.     

Cloning and characterization of trypsin- and chymotrypsin-like proteases from the midgut of the sand fly vector Phlebotomus papatasi

Trypsin and chymotrypsin serine proteases are the main digestive proteases in Diptera midguts and are also involved in many aspects of the vector-parasite relationship. In sand flies, these proteases have been shown to be a potential barrier to Leishmania growth and development within the midgut. Here we describe the sequence and partial characterization of six Phlebotomus papatasi midgut serine proteases: two chymotrypsin-like (Ppchym1 and Ppchym2) and four trypsin-like (Pptryp1-Pptryp4). All six enzymes show structural features typical to each type, including the histidine, aspartic acid, and serine (H/D/S) catalytic triad, six conserved cysteine residues, and other amino acid residues involved in substrate specificity. They also show a high degree of homology (40-60% identical residues) with their counterparts from other insect vectors, such as Anopheles gambiae and Aedes aegypti. The mRNA expression profiles of these six proteases vary considerably: two trypsin-like proteases (Pptryp1 and Pptryp2) are downregulated and one (Pptryp4) upregulated upon blood feeding. The two chymotrypsin-like enzymes display expression behavior similar to that of the early and late trypsins from Ae. aegypti.     

Quantum chemical study of the "catalytic triad" of serine proteases

Using the semi-empirical MNDO/H method several systems simulating the reaction of tetrahedral intermediate formation in the active site of serine proteases have been studied. The role played by elements of the "catalytic triad" in increasing the reactivity of serine hydroxyl has been discussed. The formation of a strong hydrogen bond between His and Asp was shown to be important in lowering the activation energy in the reaction of Ser with substrate. The change in position of the proton located between Ser and His and between His and Asp was analysed. The influence of substrate distortion on the energy of intermediate formation has been considered.     

A theoretical study of the active sites of papain and S195C rat trypsin: implications for the low reactivity of mutant serine proteinases.

The serine and cysteine proteinases represent two important classes of enzymes that use a catalytic triad to hydrolyze peptides and esters. The active site of the serine proteinases consists of three key residues, Asp...His...Ser. The hydroxyl group of serine functions as a nucleophile and the imidazole ring of histidine functions as a general acid/general base during catalysis. Similarly, the active site of the cysteine proteinases also involves three key residues: Asn, His, and Cys. The active site of the cysteine proteinases is generally believed to exist as a zwitterion (Asn...His+...Cys-) with the thiolate anion of the cysteine functioning as a nucleophile during the initial stages of catalysis. Curiously, the mutant serine proteinases, thiol subtilisin and thiol trypsin, which have the hybrid Asp...His...Cys triad, are almost catalytically inert. In this study, ab initio Hartree-Fock calculations have been performed on the active sites of papain and the mutant serine proteinase S195C rat trypsin. These calculations predict that the active site of papain exists predominately as a zwitterion (Cys-...His+...Asn). However, similar calculations on S195C rat trypsin demonstrate that the thiol mutant is unable to form a reactive thiolate anion prior to catalysis. Furthermore, structural comparisons between native papain and S195C rat trypsin have demonstrated that the spatial juxtapositions of the triad residues have been inverted in the serine and cysteine proteinases and, on this basis, I argue that it is impossible to convert a serine proteinase to a cysteine proteinase by site-directed mutagenesis.     

Identification and characterisation of the catalytic triad of the alkaliphilic thermotolerant PHA depolymerase PhaZ7 of Paucimonas lemoignei

The recently discovered extracellular poly[(R)-3-hydroxybutyrate] (PHB) depolymerase PhaZ7 of Paucimonas lemoignei represents the first member of a new subgroup (EC 3.1.1.75) of serine hydrolases with no significant amino acid similarities to conventional PHB depolymerases, lipases or other hydrolases except for a potential lipase box-like motif (Ala-His-Ser136-Met-Gly) and potential candidates for catalytic triad and oxyanion pocket amino acids. In order to identify amino acids essential for activity 11 mutants of phaZ7 were generated by site-directed mutagenesis and expressed in recombinant protease-deficient Bacillus subtilis WB800. The wild-type depolymerase and 10 of the 11 mutant proteins (except for Ser136Cys) were expressed and efficiently secreted by B. subtilis as shown by Western blots of cell-free culture fluid proteins. No PHB depolymerase activity was detected in strains harbouring one of the following substitutions: His47Ala, Ser136Ala, Asp242Ala, Asp242Asn, His306Ala, indicating the importance of these amino acids for activity. Replacement of Ser136 by Thr resulted in a decrease of activity to about 20% of the wild-type level and suggested that the hydroxy group of the serine side chain is important for activity but can be partially replaced by the hydroxy function of threonine. Alterations of Asp256 to Ala or Asn or of the putative serine hydrolase pentapeptide motif (Ala-His-Ser136-Met-Gly) to a lipase box consensus sequence (Gly134-His-Ser136-Met-Gly) or to the PHB depolymerase box consensus sequence (Gly134-Leu135-Ser136-Met-Gly) had no significant effect on PHB depolymerase activity, indicating that these amino acids or sequence motifs were not essential for activity. In conclusion, the PHB depolymerase PhaZ7 is a serine hydrolase with a catalytic triad and oxyanion pocket consisting of His47, Ser136, Asp242 and His306.     

N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases.

Recently we tentatively identified, by sequence comparison, central domains of the NS3 proteins of flaviviruses and the respective portion of the pestivirus polyprotein as RNA helicases (A.E.G. et al., submitted). Alignment of the N-proximal domains of the same proteins revealed conservation of short sequence stretches resembling those around the catalytic Ser, His and Asp residues of chymotrypsin-like proteases. A statistically significant similarity has been detected between the sequences of these domains and those of the C-terminal serine protease domains of alphavirus capsid proteins. It is suggested that flavivirus NS3 and the respective pestivirus protein contain at least two functional domains, the N-proximal protease and the C-proximal helicase one. The protease domain is probably involved in the processing of viral non-structural proteins.     

Cloning and characterization of chymotrypsin- and trypsin-like cDNAs from the gut of the Hessian fly [Mayetiola destructor (Say)

Fifteen unique cDNA clones encoding trypsin- or chymotrypsin-like proteins were cloned and characterized from a gut cDNA library derived from Hessian fly [Mayetiola destructor (Say)] larvae. Based on sequence similarities, the cDNAs were sorted into five gene groups, which were named MDP1 to MDP5. Two of the gene groups, MDP1 and MDP2, encoded chymotrypsin-like proteins; the other three encoded putative trypsins. All deduced proteins have conserved His(87), Asp(136), and Ser(241) residues for the catalytic triad and three pairs of cysteine residues for disulfide bridge configurations. The substrate specificity determination residue at position 235 was also conserved in the putative trypsins and chymotrypsins. In addition, all the deduced protein precursors had a typical secretion signal peptide and activation peptide. Northern blot analysis revealed that all these gene groups were exclusively expressed in the larval stage. The expression profiles for each gene group differed significantly in different ages of the larva, as well as in different tissues. Protease activity analysis of gut extract, using specific inhibitors, demonstrated that serine proteases were the major digestive enzymes in the gut of M. destructor larvae. Serine protease inhibitors inhibited as much as 90% proteolytic activities of gut extract, whereas inhibitors specific to other proteases, including cysteine proteases, aspartic proteases, and metallo-proteases, inhibited only 10-24% of gut protease activity.     

Identification and analysis of a new family of bacterial serine proteinases

A family of hypothetical proteins, identified predominantly from archaeal genomes, has been analyzed in order to understand its functional characteristics. Using extensive sequence similarity searches it is inferred that this family is remotely related (best sequence identity is 19%) to ClpP proteinases that belongs to serine proteinase class. This family of hypothetical proteins is referred to as SDH proteinase family based on conserved sequential order of Ser, Asp and His residues and predicted serine proteinase activity. Results of fold recognition of SDH family sequences confirmed the remote relationship between SDH proteinases and Clp proteinases and revealed similar tertiary location of putative catalytic triad residues critical for serine proteinase function. However, the best sequence alignment we could obtain suggests that while catalytic Ser is conserved across Clp and SDH proteinases the location of the other catalytic triad residues, namely, His and Asp are swapped in their amino acid alignment positions and hence in 3-D structure. The evidence of conserved catalytic triad suggests that SDH could be a new family of serine proteinases with the fold of Clp proteinase, however sharing the catalytic triad order of carboxypeptidase clan. Signal peptide sequence identified at the N-terminus of some of the homologues suggests that these might be secretory serine proteinases involved in cleavage of extracellular proteins while the remote homologues, ClpP proteinases, are known to work in intracellular environment.     

Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin-like specificities

The three-dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4A resolution. The enzyme, which has both trypsin-like and chymotrypsin-like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin-like and chymotrypsin-like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities.     

Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface

Lc-WT, the wild-type light chain of antibody, and Lc-Triad, its double mutant with E1D and T27aS designing for the construction of catalytic triad within Asp1, Ser27a, and original His93 residues, were displayed on the cell surface of the protease-deficient yeast strain BJ2168. When each cell suspension was reacted with BODIPY FL casein and seven kinds of peptide-MCA substrates, respectively, a remarkable difference in hydrolytic activities toward Suc-GPLGP-MCA (succinyl-Gly-Pro-Leu-Gly-Pro-MCA), a substrate toward collagenase-like peptidase, was observed between the constructs: Lc-Triad-displaying cells showed higher catalytic activity than Lc-WT-displaying cells. The difference disappeared in the presence of the serine protease inhibitor diisopropylfluorophosphate, suggesting that the three amino acid residues, Ser27a, His93, and Asp1, functioned as a catalytic triad responsible for the proteolytic activity in a similar way to the anti-vasoactive intestinal peptide (VIP) antibody light chain. A serine protease-like catalytic triad (Ser, His, and Asp) is considered to be directly involved in the catalytic mechanism of the anti-VIP antibody light chain, which moderately catalyzes the hydrolysis of VIP. These results suggest the possibility of new approach for the creation of tailor-made proteases beyond limitations of the traditional immunization approach.     

Chlorophyllase as a serine hydrolase: identification of a putative catalytic triad

Chlorophyllases (Chlases), cloned so far, contain a lipase motif with the active serine residue of the catalytic triad of triglyceride lipases. Inhibitors specific for the catalytic serine residue in serine hydrolases, which include lipases effectively inhibited the activity of the recombinant Chenopodium album Chlase (CaCLH). From this evidence we assumed that the catalytic mechanism of hydrolysis by Chlase might be similar to those of serine hydrolases that have a catalytic triad composed of serine, histidine and aspartic acid in their active site. Thus, we introduced mutations into the putative catalytic residue (Ser162) and conserved amino acid residues (histidine, aspartic acid and cysteine) to generate recombinant CaCLH mutants. The three amino acid residues (Ser162, Asp191 and His262) essential for Chlase activity were identified. These results indicate that Chlase is a serine hydrolase and, by analogy with a plausible catalytic mechanism of serine hydrolases, we proposed a mechanism for hydrolysis catalyzed by Chlase.     

Contribution of active site residues to substrate hydrolysis by USP2: insights into catalysis by ubiquitin specific proteases

The ubiquitin-specific protease (USP) structural class represents the largest and most diverse family of deubiquitinating enzymes (DUBs). Many USPs assume important biological roles and emerge as potential targets for therapeutic intervention. A clear understanding of USP catalytic mechanism requires a functional evaluation of the proposed key active site residues. Crystallographic data of ubiquitin aldehyde adducts of USP catalytic cores provided structural details on the catalytic triad residues, namely the conserved Cys and His, and a variable putative third residue, and inferred indirect structural roles for two other conserved residues (Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of the tetrahedral intermediate (TI). We have expressed the catalytic domain of USP2 and probed by site-directed mutagenesis the role of these active site residues in the hydrolysis of peptide and isopeptide substrates, including a synthetic K48-linked diubiquitin substrate for which a label-free, mass spectrometry based assay has been developed to monitor cleavage. Hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecular dynamics simulations of USP2, free and complexed with the TI of a bona fide isopeptide substrate, were carried out. We found that Asn271 is structurally poised to directly stabilize the oxyanion developed in the acylation step, while being structurally supported by the adjacent absolutely conserved Asp575. Mutagenesis data functionally confirmed this structural role independent of the nature (isopeptide vs peptide) of the bond being cleaved. We also found that Asn574, structurally located as the third member of the catalytic triad, does not fulfill this role functionally. A dual supporting role is inferred from double-point mutation and structural data for the absolutely conserved residue Asp575, in oxyanion hole formation, and in maintaining the correct alignment and protonation of His557 for catalytic competency.     

Modeling and structural analysis of evolutionarily diverse S8 family serine proteases

Serine proteases are an abundant class of enzymes that are involved in a wide range of physiological processes and are classified into clans sharing structural homology. The active site of the subtilisin-like clan contains a catalytic triad in the order Asp, His, Ser (S8 family) or a catalytic tetrad in the order Glu, Asp and Ser (S53 family). The core structure and active site geometry of these proteases is of interest for many applications. The aim of this study was to investigate the structural properties of different S8 family serine proteases from a diverse range of taxa using molecular modeling techniques. In conjunction with 12 experimentally determined three-dimensional structures of S8 family members, our predicted structures from an archaeon, protozoan and a plant were used for analysis of the catalytic core. Amino acid sequences were obtained from the MEROPS database and submitted to the LOOPP server for threading based structure prediction. The predicted structures were refined and validated using PROCHECK, SCRWL and MODELYN. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S8 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, the analysis presented here is beneficial for future molecular modeling strategies and structure-based rational drug design.