Isolation and characterization of fibrinogenase from western diamondback rattlesnake venom and its comparison to the thrombin-like enzyme, crotalase

A new type of fibrinogenase was isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Unlike thrombin, the newly isolated fibrinogenase did not cause formation of a fibrin clot. Various properties of the fibrinogenase we isolated were compared with crotalase isolated from the venom of C. adamanteus. It was found that fibrinogenase has considerable similarity to crotalase isolated by Markland and Damus in 1971. Crotalase is a thrombin-like enzyme and produces a fibrin clot from fibrinogen. The A alpha chain of fibrinogen was first split and the B beta chain was cleaved later. The fact that no fibrin clot forms indicates that the cleavage sites in A alpha and B beta chains of fibrinogen must be different from thrombin sites. The fibrinogenase also released bradykinin by interacting with plasma proteins. It hydrolyzed TAME (p-toluenesulfonyl-L-arginine methyl ester), BAEE (N-benzoyl-L-arginine ethyl ester). TLME (N-tosyllysine methyl ester) but not BAA (N-benzoylarginine amide), TAA (N-tosylarginineamide) or ATEE (N-acetyltyrosine ethyl ester). The enzyme is an acidic protein with pI of 4.6 and a mol. wt of 31,000. It consists of 272 total amino acid residues, 21% of which are acidic amino acids. Fibrinogenase is a specific form of protease. A newly liberated amino group after hydrolysis of dimethyl-casein can be detected by the reagent trinitrobenzenesulfonic acid (TNBS). Fibrinogenase differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural features of a snake venom thrombin-like enzyme: thrombin and trypsin on a single catalytic platform?

The Lachesis muta thrombin-like enzyme (LM-TL) is a single chain serine protease that shares 38% sequence identity with the serine protease domain of thrombin and also displays similar fibrinogen-clotting activity. In addition, the 228 amino acid residue LM-TL is 52% identical to trypsin, and cleaves chromogenic substrates with similar specificity. Herein we report a three-dimensional (3D) model validated experimentally for LM-TL based on these two homologous proteins of known 3D structure. Spatial modeling of LM-TL reveals a serine protease with a chymotrypsin fold presenting a hydrophobic pocket on its surface, involved in substrate recognition, and an important 90's loop, involved in restricting the LM-TL catalytic site cleft. Docking analysis showed that LM-TL would not form a stable complex with basic pancreatic trypsin inhibitor and wild-type ecotin since its 90's loop would restrict the access to the catalytic site. LM-TL formed acceptable interactions with fibrinopeptide A and a variant of ecotin; ecotin-TSRR/R in which both the primary and secondary binding sites are mutated Val81Thr, Thr83Ser, Met84Arg, Met85Arg and Asp70Arg. Furthermore, analysis of the primary structures of LM-TL and of the seven snake venom thrombin-like enzymes (SVTLEs) family reveals a subgroup formed by LM-TL, crotalase, and bilineobin, both closely related to thrombin. Therefore, LM-TL provides an initial point to compare SVTLEs with their counterparts, e.g. the mammalian serine proteases, and a basis for the localization of important residues within the little known SVTLEs family.     

The primary structure of crotalase,a thrombin-like venom enzyme,exhibits closer homology to kallikrein than to other serine proteases

Amino acid sequences of crotalase, totaling 98 residues or about 37% of the molecule, were determined by Edman degradation and were compared with the published sequences of nine serine proteases. Homologous alignment could be found for all crotalase sequences except one decapeptide. Comparison between crotalase and porcine pancreatic kallikrein showed the largest number of identical amino acids (36%). This finding has led to experiments which demonstrate that crotalase has specific enzymatic properties resembling kallikrein.     

Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi

HtrA2/Omi, a mitochondrial serine protease in mammals, is important in programmed cell death. However, the underlining mechanism of HtrA2/Omi-mediated apoptosis remains unclear. Analogous to the bacterial homolog HtrA (DegP), the mature HtrA2 protein contains a central serine protease domain and a C-terminal PDZ domain. The 2.0 A crystal structure of HtrA2/Omi reveals the formation of a pyramid-shaped homotrimer mediated exclusively by the serine protease domains. The peptide-binding pocket of the PDZ domain is buried in the intimate interface between the PDZ and the protease domains. Mutational analysis reveals that the monomeric HtrA2/Omi mutants are unable to induce cell death and are deficient in protease activity. The PDZ domain modulates HtrA2/Omi-mediated cell death activity by regulating its serine protease activity. These structural and biochemical observations provide an important framework for deciphering the mechanisms of HtrA2/Omi-mediated apoptosis.     

Is histoaspartic protease a serine protease with a pepsin‐like fold?

The primary structure of the so-called histoaspartic protease from Plasmodium falciparum has a very high percentage of identity and homology with the pepsin-like enzyme plasmepsin II. A homology modeling approach was used to calculate the three-dimensional structure of the enzyme. Molecular dynamics (MD) simulations were applied to find those structural properties of the histoaspartic protease that had a tendency to remain stable during all runs. The results have shown that hydrogen-bonded residues Ser37-His34-Asp214 are arranged without any strain, in a manner that resembles the active site of a serine protease, while Ser38 and Asn39 take up positions appropriate to formation of an oxyanion hole. Although there are several important differences between the enzyme and plasmepsin II, all of the structural features associated with a typical pepsin-like aspartic protease are present in the final model of the histoaspartic protease. A possibility that this enzyme may function as a serine protease is discussed.     

绿木霉Gv29-8丝氨酸蛋白酶S8/S53超家族基因特性及功能

【背景】丝氨酸蛋白酶在木霉菌生物防治过程中发挥重要作用。【目的】研究绿木霉丝氨酸蛋白酶S8/S53超家族基因信息及其生物学功能,进而为该蛋白酶生防制剂的开发及基因改造提供理论支持。【方法】通过生物信息学分析方法,从绿木霉Gv29-8基因组中鉴定出23个丝氨酸蛋白酶基因,以少孢节丛孢菌ATCC 24927基因组中鉴定的4个丝氨酸蛋白酶基因作为对照,对这27个丝氨酸蛋白酶基因的特性、蛋白结构、进化地位、功能等进行预测分析。【结果】27个基因结构差异较大,编码的蛋白具有典型的丝氨酸蛋白酶催化三联体结构,属于S8/S53超家族,分为6个亚家族,同一亚家族的蛋白酶保守区长度相近,相似性较高,催化残基附近序列比较保守。系统进化分析显示,同一亚家族丝氨酸蛋白酶聚为一类。【结论】绿木霉和少孢节丛孢菌的部分丝氨酸蛋白酶基因在结构和蛋白性质上相似性强,亲缘关系较近,均属于S8_PCSK9_ProteinaseK_like亚家族,推测绿木霉与少孢节丛孢菌该亚家族的丝氨酸蛋白酶具有相似的功能,可抑制植物病原真菌和降解线虫体壁。     

Mechanistic role of NS4A and substrate in the activation of HCV NS3 protease

Hepatitis C virus (HCV) NS3 protease is the key enzyme for its maturation. Three hypotheses have been advanced in the literature to demonstrate the mechanism of the activation of the HCV NS3 protease. A virus-encoded protein NS4A and substrate are proposed to be involved in the activation of the HCV NS3 protease. However, the three hypotheses are not completely consistent with one another. Multiple molecular dynamics simulations were performed on various NS3 protease systems: free NS3 protease, NS3/4A, NS3/inhibitor, and NS3/4A/inhibitor complexes, to further unravel the mechanism of the activation of the NS3 protease. Simulation results suggest that the binding of NS4A induces a classic serine protease conformation of the catalytic triad of the NS3 protease. NS4A rearranges the secondary structure of both the N-terminus and catalytic site of the NS3 protease, reduces the mobility of the global structure of the NS3 protease, especially the catalytic site, and provides a rigid and tight structure, except for the S1 pocket, for the binding and hydrolysis of substrates. The binding of substrate also contributes to the activation of the NS3 protease by an induced-fit of the classic serine protease catalytic triad. However, the global structure of the NS3 protease is still loose and highly flexible without stable secondary structural elements, such as helix α0 at the N-terminus and helix α1 and β-sheet E1-F1 at the catalytic site. The structure of the NS3 protease without NS4A is not suitable for the binding and hydrolysis of substrates.     

Crystal structure of a supercharged variant of the human enteropeptidase light chain

The highly specific serine protease human enteropeptidase light chain cleaves the Asp4Lys recognition sequence and represents an interesting enzyme for biotechnological applications. The human enzyme shows 10 times faster kinetics compared to other animal sources but low solubility under low salt conditions, which hampers protein production and crystallization. Therefore, a supercharged variant (N6D/G21D/G22D/N142D/K210E/C112S) with increased solubility was used for crystallization. The structure (resolution, 1.9 Å) displays a typical α/β trypsin‐like serine protease‐fold. The mutations introduced for protein supercharging generate larger clusters of negative potential on both sites of the active cleft but do not affect the structural integrity of the protein. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen.

Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.     

Crystal structure of a novel viral protease with a serine/lysine catalytic dyad mechanism

The blotched snakehead virus (BSNV), an aquatic birnavirus, encodes a polyprotein (NH2-pVP2-X-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease (VP4) to liberate itself and the viral proteins pVP2, X and VP3. The protein pVP2 is further processed by VP4 to give rise to the capsid protein VP2 and four structural peptides. We report here the crystal structure of a VP4 protease from BSNV, which displays a catalytic serine/lysine dyad in its active site. This is the first crystal structure of a birnavirus protease and the first crystal structure of a viral protease that utilizes a lysine general base in its catalytic mechanism. The topology of the VP4 substrate binding site is consistent with the enzymes substrate specificity and a nucleophilic attack from the si-face of the substrates scissile bond. Despite low levels of sequence identity, VP4 shows similarities in its active site to other characterized Ser/Lys proteases such as signal peptidase, LexA protease and Lon protease. Together, the structure of VP4 provides insights into the mechanism of a recently characterized clan of serine proteases that utilize a lysine general base and reveals the structure of potential targets for antiviral therapy, especially for other related and economically important viruses, such as infectious bursal disease virus in poultry and infectious pancreatic necrosis virus in aquaculture.     

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.     

丙型肝炎病毒丝氨酸蛋白酶特异性结合肽的筛选和鉴定

丙型肝炎病毒丝氨酸蛋白酶在病毒复制和包装中的重要作用使其成为特异性抗病毒药物研究的首选靶标。根据丝氨酸蛋白酶晶体结构特点,用柔性连接子连接NS3丝氨酸蛋白酶结构域和NS4A的核心序列,构建成单链丝氨酸蛋白酶基因并且在大肠杆菌中获得高水平的可溶性表达,纯化后的目的蛋白能够切割重组蛋白底物NS5ab。随后,以单链丝氨酸蛋白酶为靶分子对噬菌体展示的随机十二肽库进行了三轮淘筛,挑选的44个克隆中有37个克隆能够特异性地结合丝氨酸蛋白酶,并且这种结合作用为竞争性ELISA试验结果所支持。对13个克隆进行序列测定,得到6种序列,它们在氨基酸组成上存在明显偏性,富含组氨酸和色氨酸,缺乏酸性氨基酸;6种序列存在一个共有序列。     

NMR structure determination of tick anticoagulant peptide (TAP).

Tick anticoagulant peptide (TAP) is a potent and selective 60-amino acid inhibitor of the serine protease Factor Xa (fXa), the penultimate enzyme in the blood coagulation cascade. The structural features of TAP responsible for its remarkable specificity for fXa are unknown, but the binding to its target appears to be unique. The elucidation of the TAP structure may facilitate our understanding of this new mode of serine protease inhibition and could provide a basis for the design of novel fXa inhibitors. Analyses of homo- and heteronuclear two-dimensional NMR spectra (total correlation spectroscopy, nuclear Overhauser effect spectroscopy [NOESY], constant time heteronuclear single quantum correlation spectroscopy [CT-HSQC], and HSQC-NOESY; 600 MHz; 1.5 mM TAP; pH 2.5) of unlabeled, 13C-labeled, and 15N-labeled TAP provided nearly complete 1H sequence-specific resonance assignments. Secondary structural elements were identified by characteristic NOE patterns and D2O amide proton-exchange experiments. A three-dimensional structure of TAP was generated from 412 NOESY-derived distance and 47 dihedral angle constraints. The structural elements of TAP are similar in some respects to those of the Kunitz serine protease inhibitor family, with which TAP shares weak sequence homology. This structure, coupled with previous kinetic and biochemical information, confirms previous suggestions that TAP has a unique mode of binding to fXa.     

Conformational similarities between one-chain and two-chain tissue plasminogen activator (t-PA): implications to the activation mechanism on one-chain t-PA.

Tissue plasminogen activator (t-PA) is an exceptional serine protease, because unlike most other serine protease zymogens single-chain tissue plasminogen activator (sct-PA) possesses a substantial amount of proteolytic activity. The unusual reaction of sct-PA afforded the opportunity to directly compare the active site environment of sct-PA and two-chain tissue plasminogen activator (tct-PA) in solution through the application of a series of nitroxide spin labels and fluorophores. These labels, which have been previously shown to covalently label the catalytic serine of other serine proteases, inactivated both sct-PA and tct-PA. The labels can be divided into two classes: those which form tetrahedral complexes (sulfonates) and those which form trigonal complexes (anthranilates). Those which formed tetrahedral complexes were found to be insensitive to structural differences between sct-PA and tct-PA at the active site. In contrast, those which formed trigonal complexes could differentiate and monitor the sct-PA to tct-PA conversion by fluorescence spectroscopy. Models of the structure of sct-PA and tct-PA were constructed on the basis of the known X-ray structures of other serine protease zymogen and active enzyme forms. One of the nitroxide spin labels was modeled into the sct-PA and tct-PA structures in two possible orientations, both of which could be sensitive to structural differences between sct-PA and tct-PA. These models formed the structural rationale used to explain the results obtained with the "tetrahedral" and "trigonal" probes, as well as to offer a possible explanation for the unique reactivity of sct-PA.     

Exploring structurally conserved solvent sites in protein families

Protein-bound water molecules are important components of protein structure, and therefore, protein function and energetics. Although structural conservation of solvent has been studied in a few protein families, a lack of suitable computational tools has hindered more comprehensive analyses. Herein we present a semiautomated computational approach for identifying solvent sites that are conserved among proteins sharing a common three-dimensional structure. This method is tested on six protein families: (1) monodomain cytochrome c, (2) fatty-acid binding protein, (3) lactate/malate dehydrogenase, (4) parvalbumin, (5) phospholipase A2, and (6) serine protease. For each family, the method successfully identified previously known conserved solvent sites. Moreover, the method discovered 22 novel conserved solvent sites, some of which have higher degrees of conservation than the previously known sites. All six families studied had solvent sites with more than 90% conservation and these sites were invariably located in regions of the protein with very high sequence conservation. These results suggest that highly conserved solvent sites, by virtue of their proximity to conserved residues, should be considered as one of the defining three-dimensional structural characteristics of protein families and folds.     

Dengue virus NS3 serine protease. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects

The mosquito-borne dengue viruses are widespread human pathogens causing dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, placing 40% of the world's population at risk with no effective treatment. The viral genome is a positive strand RNA that encodes a single polyprotein precursor. Processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by NS3 serine protease, which requires NS2B as a cofactor. We report here the crystal structure of the NS3 serine protease domain at 2.1 A resolution. This structure of the protease combined with modeling of peptide substrates into the active site suggests identities of residues involved in substrate recognition as well as providing a structural basis for several mutational effects on enzyme activity. This structure will be useful for development of specific inhibitors as therapeutics against dengue and other flaviviral proteases.     

Common protein architecture and binding sites in proteases utilizing a Ser/Lys dyad mechanism

Escherichia coli signal peptidase (SPase) and E. coli UmuD protease are members of an evolutionary clan of serine proteases that apparently utilize a serine-lysine catalytic dyad mechanism. Recently, the crystallographic structure of a SPase inhibitor complex was solved elucidating the catalytic residues and the substrate binding subsites. Here we show a detailed comparison of the E. coli SPase structure to the native E. coli UmuD' structure. The comparison reveals that despite a very low sequence identity these functionally diverse enzymes share the same protein fold within their catalytic core and allows by analogy for the assignment of the cleavage-site orientation and substrate binding subsites in the UmuD(D') protease. The structural alignment of SPase and UmuD' predicts important mechanistic and structural similarities and differences within these newly characterized families of serine proteases.     

新型冠状病毒相关TMPRSS2蛋白结构特征和抗原表位分析

采用生物信息学方法分析预测新型冠状病毒(Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)跨膜蛋白酶丝氨酸2(transmembrane protease serine 2, TMPRSS2)的理化特性、结构特征和抗原表位,为抗SARS-CoV-2药物研发提供参考。利用ProtParam、ProtScale分析预测TMPRSS2蛋白酶的理化特性;利用COILS Server、SignalP、TMPred、TargetP Server、NetPhos Server、NetNGlyc Server服务器对TMPRSS2蛋白酶结构进行功能结构的分析预测;利用SOPMA、Pfam、SWISS-MODEL分析预测TMPRSS2蛋白酶高级结构;利用IEBD分析预测TMPRSS2蛋白酶B细胞、T细胞表位。TMPRSS2蛋白酶氨基酸组成数为492个,其中丝氨酸占比最高;亲水性较高,含10个跨膜螺旋区;具有4个磷酸化位点,3个糖基化修饰点;二级结构中无规则卷曲占据主导地位,三级结构能与已知的5ce.1.1.A(SMTL ID)模型同源建模;存在13个潜在的B细胞表位,12个得分较高的T细胞表位。     

Expression, crystallization, and three-dimensional structure of the catalytic domain of human plasma kallikrein

Plasma kallikrein is a serine protease that has many important functions, including modulation of blood pressure, complement activation, and mediation and maintenance of inflammatory responses. Although plasma kallikrein has been purified for 40 years, its structure has not been elucidated. In this report, we described two systems (Pichia pastoris and baculovirus/Sf9 cells) for expression of the protease domain of plasma kallikrein, along with the purification and high resolution crystal structures of the two recombinant forms. In the Pichia pastoris system, the protease domain was expressed as a heterogeneously glycosylated zymogen that was activated by limited trypsin digestion and treated with endoglycosidase H deglycosidase to reduce heterogeneity from the glycosylation. The resulting protein was chromatographically resolved into four components, one of which was crystallized. In the baculovirus/Sf9 system, homogeneous, crystallizable, and nonglycosylated protein was expressed after mutagenizing three asparagines (the glycosylation sites) to glutamates. When assayed against the peptide substrates, pefachrome-PK and oxidized insulin B chain, both forms of the protease domain were found to have catalytic activity similar to that of the full-length protein. Crystallization and x-ray crystal structure determination of both forms have yielded the first three-dimensional views of the catalytic domain of plasma kallikrein. The structures, determined at 1.85 A for the endoglycosidase H-deglycosylated protease domain produced from P. pastoris and at 1.40 A for the mutagenically deglycosylated form produced from Sf9 cells, show that the protease domain adopts a typical chymotrypsin-like serine protease conformation. The structural information provides insights into the biochemical and enzymatic properties of plasma kallikrein and paves the way for structure-based design of protease inhibitors that are selective either for or against plasma kallikrein.     

An in silico approach to understand the structure–function properties of a serine protease (Bacifrinase) from Bacillus cereus and experimental evidence to support the interaction of Bacifrinase with fibrinogen and thrombin

Microbial fibrinogenolytic serine proteases find therapeutic applications in the treatment of thrombosis- and hyperfibrinogenemia-associated disorders. However, analysis of structure–function properties of an enzyme is utmost important before its commercial application. In this study, an attempt has been made to understand the structure of a fibrinogenolytic protease enzyme, “Bacifrinase” from Bacillus cereus strain AB01. From the molecular dynamics trajectory analysis, the modelled three-dimensional structure of the protease was found to be stable and the presence of a catalytic triad made up of Asp102, His83 and Ser195 suggests that it is a serine protease. To understand the mechanism of enzyme–substrate and enzyme–inhibitor interactions, the equilibrated protein was docked with human fibrinogen (the physiological substrate of this enzyme), human thrombin and with ten selective protease inhibitors. The Bacifrinase–chymostatin interaction was the strongest among the selected protease inhibitors. The serine protease inhibitor phenyl methane sulphonyl fluoride was found to interact with the Ser134 residue of Bacifrinase. Furthermore, protein–protein docking study revealed the fibrinogenolytic property of Bacifrinase and its interaction with Aα-, Bβ- and Cγ-chains human fibrinogen to a different extent. However, biochemical analysis showed that Bacifrinase did not hydrolyse the γ-chain of fibrinogen. The in silico and spectrofluorometric studies also showed interaction of Bacifrinase with thrombin as well as fibrinogen with a Kd value of 16.5 and .81 nM, respectively. Our findings have shed light on the salient structural features of Bacifrinase and confirm that it is a fibrinogenolytic serine protease.