Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence.

Proteinase B (PrB) is a subtilisin-like serine protease found in the vacuole of the yeast Saccharomyces cerevisiae. It is first made as a large precursor that consists of a putative signal sequence, a 260-amino acid pro region, the serine protease domain, and two small COOH-terminal post regions (Moehle, C. M., Dixon, C. K., and Jones, E. W. (1989) J. Cell Biol. 108, 309-324). This precursor is glycosylated and proteolytically processed at least three times before mature enzyme is formed. To determine whether an intact PrB catalytic site is required for proteolytic processing of the precursor, point mutations were generated at the codons for the active site serine or aspartate residues by site-directed mutagenesis. The effect of these mutations on PrB processing suggests that the large pro region may be cleaved by an intramolecular, autocatalytic mechanism. The properties of a prb1 mutant that accumulates a 37-kDa precursor in addition to mature sized mutant PrB antigen suggests that the final proteolytic cleavage step is also autocatalytic. A prb1 deletion that lacks codons for the large pro region was made to test whether this part of the precursor is required for formation of mature PrB. Analysis of this mutant revealed two functions for this region: it prevents N-linked glycosylation of the serine protease domain and it allows the PrB precursor to be processed by proteinase A. The pro region can fulfill this latter function if added as a separate molecule, so long as glycosylation of the catalytic domain is prevented by other means.     

Mutations at the putative junction sites of the yeast VMA1 protein, the catalytic subunit of the vacuolar membrane H(+)-ATPase, inhibit its processing by protein splicing.

A single gene, VMA1, encodes the 69-kDa subunit of the vacuolar membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae. We have proposed that the subunit is synthesized as a precursor of 120 kDa (1,071 amino acids) and then converted to the 69-kDa form by an unusual processing reaction, which removes the internal domain of 454 amino acids (residues 284-737) and joins the N- and C-terminal domains. Cysteine to serine mutations at residues 284 and 738, the residues that bracket the internal domain, were introduced into the VMA1 gene by site-directed mutagenesis, and the mutant genes were expressed in a null vma1 mutant. Cells harboring either of the mutant vma1 genes accumulate nonfunctional fragments of the subunit. The mutation of Cys-284 inhibited the cleavage of the N-terminal junction site. Cys-738-->Ser mutation appeared to block the processing at both junction sites although the mutant gene yielded a small fraction of the functional 69-kDa subunit.     

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.     

The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity.     

In vitro studies of cross-resistance mutations against two hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.     

Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis.

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.     

可视化突变建模、定点突变和表达超耐热菌D-乳酸脱氢酶

目的 构建产天然防腐剂苯乳酸的工程菌。方法 分析超耐热菌（Aquifex aeolicus,A.aeolicus ）D-乳酸脱氢酶（D-LDH）的三维构象,并与构建的可视化突变体三维模型进行对比,通过比较酶活性中心氨基酸残基与底物的空间构象,优选最佳模型进行定点突变,克隆、表达和苯乳酸发酵实验。结果 优选到F49A和Y297S两个单突变模型和一个F49A/Y297S双突变模型;分别进行定点突变和工程菌构建,三个突变工程菌,均能发酵产生苯乳酸。结论 可视化定点突变乳酸脱氢酶可作为构建高产苯乳酸工程菌的有效方法。     

Primary site and second site revertants of missense mutants of the evolutionarily invariant tryptophan 64 in iso-1-cytochrome c from yeast.

The three missense mutants cyc1-132, cyc1-166 and cyc1-189 in the yeast Saccharomyces cerevisiae contain nonfunctional and thermolabile iso-1-cytochromes c and have different replacements of the tryptophan at position 64 which corresponds to the invariant tryptophan residue found in cytochromes c from all eukaryotic species. The cyc1-166 and cyc1-189 mutants contain single replacements of, respectively, serine 64 and cysteine 64, while the cyc1-132 mutant contains a double replacement of glycine 64 and alanine 65 instead of the normal tryptophan 64 and aspartic acid 65. Twenty-three intragenic revertants having at least partially functional iso-1-cytochromes c arose from these three missense mutants by single amino acid replacements of either tryptophan, phenylalanine, tyrosine or leucine at position 64, or by second-site replacements in which the mutant residues at position 64 are retained and the normal serine 45 is replaced by phenylalanine 45. Specific activities of the iso-1-cytochromes c were estimated by growth of strains on lactate medium and are as follows, in terms of the normal, for iso-1-cytochromes c altered specifically in the ways shown: 100% for phenylalanine 64; 25% for tyrosine 64; between 0 and 25% for leucine 64; 100% for phenylalanine 45, cysteine 64; 25% for phenylalanine 45, serine 64; between 0 and 25% for phenylalanine 45, glycine 64, alanine 65; and 0% for serine 64, for cysteine 64, and for glycine 64, alanine 65 iso-1-cytochromes c. The results demonstrate that small residues of glycine, serine, and cysteine at position 64 are incompatible with function; they imply that many of the 10 amino acids accessible by single base-pair substitution but not observed in primary site revertants also are incompatible with function; and they show that large hydrophobic residues of phenylalanine, leucine, and tyrosine at position 64 are capable of restoring at least partial function. The second site revertants indicate that deleterious effects of the three missense mutants can be compensated by the introduction of phenylalanine 45, which may occupy space normally filled by tryptophan 64. Altered shapes of Calpha-band spectra and at least partial instability were characteristics of all iso-1-cytochromes c found lacking tryptophan 64. Apparently, the principal role of the invariant tryptophan is stabilization of the active protein structure, by providing a large hydrophobic group at the proper location.     

Glycoprotein B of herpes simplex virus type 1 oligomerizes through the intermolecular interaction of a 28-amino-acid domain.

Herpes simplex virus type 1 glycoprotein B (gB) is an envelope component that plays an essential role in virus infection. The biologically active form of gB is an oligomer that contributes to the process of viral envelope fusion with the cell surface membrane, resulting in viral penetration and initiation of the replication cycle. In previous studies, two discontinuous sites for oligomer formation were identified: a nonessential upstream site located between residues 93 and 282 and an essential downstream site located between residues 596 and 711. In this study, in vitro-transcribed and -translated gB test molecules were used to characterize the more active essential membrane-proximal domain. A series of gB test polypeptides mutated in this downstream oligomerization domain were assayed for their abilities to form oligomers with a mutant gB capture polypeptide containing the analogous wild-type domain. Detection of oligomers was achieved by coimmunoprecipitation of two gB mutant molecules by using a monoclonal antibody specific for a hemagglutinin epitope tag introduced into the coding sequence of the capture polypeptide. Analysis of the immune-precipitated products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the downstream oligomerization domain resided within residues 626 to 676. This region was further resolved into two segments, residues 626 to 653 and 653 to 675, each of which was independently sufficient to form oligomers. However, residues 626 to 653 provided for a stronger interaction between gB monomers. Moreover, this stretch of 28 amino acids was shown to form oligomers when introduced into the carboxy-terminal region of gB monomers lacking this domain at the normal site, thus indicating that this domain was functionally independent of its natural location within the gB molecule. Further analysis of the sequence within residues 596 to 653 by using mutant test polypeptides altered in individual amino acids revealed that cysteines 9 and 10 located at positions 596 and 633, respectively, were not required for oligomer formation but contributed to dimer formation and/or stabilization. The results of this study suggest that oligomerization of gB monomers is induced by interactions between contiguous residues localized within the ectodomain near the site of molecule insertion into the viral envelope membrane.     

Crystal structure at 1.63 A resolution of the native form of porcine beta-trypsin: revealing an acetate ion binding site and functional water network

The active center of a serine protease is the catalytic triad composed of His-57, Ser-195 and Asp-102. The existing crystal structure data on serine proteases have not fully answered a number of fundamental questions relating to the catalytic activity of serine proteases. The new high resolution native porcine beta-trypsin (BPT) structure is aimed at extending the knowledge on the conformation of the active site and the ordered water structure within and around the active site. The crystal structure of BPT has been determined at 1.63 A resolution. An acetate ion bound at the active site of a trypsin molecule by both classical hydrogen bonds and C-HellipsisO hydrogen bonds has been identified for the first time. A large network of water molecules extending from the recognition amino acid Asp-184 to the entry of the active site has been observed in the BPT structure. A detailed comparison with inhibitor complexes and autolysates indicates that the sulfate ion and the acetate ion bind at the same site of the trypsin molecule. The Ser-195 Cbeta-Ogamma-His-57 Nepsilon angle in the catalytic triad of BPT is intermediate between the corresponding values of the complex and native structure due to acetate ion binding. The network of waters from the recognition amino acid to the active site entry is probably the first ever complete picture of functional waters around the active site. Structural comparisons show that the functional waters involved in the binding of small molecule inhibitors and protease inhibitors are distinctly different.     

Reaction of LexA repressor with diisopropyl fluorophosphate. A test of the serine protease model

The LexA repressor of Escherichia coli modulates the expression of the SOS regulon. In the presence of DNA damaging agents in vivo, the 202-amino acid LexA repressor is inactivated by specific RecA-mediated cleavage of the Ala-84/Gly-85 peptide bond. In vitro. LexA cleavage requires activated RecA at neutral pH, and proceeds spontaneously at high pH in an intramolecular reaction termed autodigestion. A model has been proposed for the mechanism of autodigestion in which serine 119 serves as the reactive nucleophile that attacks the Ala-84/Gly-85 peptide bond in a manner analogous to a serine protease, while uncharged lysine 156 activates the serine 119 hydroxyl group. In this work, we have tested this model by examining the effect of the serine protease inhibitor diisopropyl fluorophosphate (DFP) on autodigestion. We found that DFP inhibited autodigestion and that serine 119 was the only serine residue to react with DFP. We also examined [3H]DFP incorporation by a number of cleavage-impaired LexA mutant proteins and found that mutations in the proposed active site, but not in the cleavage site, significantly reduced the rate of [3H]DFP incorporation. Finally, we showed that the purified carboxyl-terminal domain, which contains the proposed catalytic residues, incorporated [3H]DFP at a rate indistinguishable from the intact protein. These data further support our current model for the mechanism of autodigestion and the organization of LexA.     

Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly

Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6(Gag) or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.     

Crystal structures of Urtica dioica agglutinin and its complex with tri-N-acetylchitotriose

Urtica dioica agglutinin is a small plant lectin that binds chitin. We purified the isolectin VI (UDA-VI) and crystal structures of the isolectin and its complex with tri-N-acetylchitotriose (NAG3) were determined by X-ray analysis. The UDA-VI consists of two domains analogous to hevein and the backbone folding of each domain is maintained by four disulfide bridges. The sequence similarity of the two domains is not high (42 %) but their backbone structures are well superimposed except some loop regions. The chitin binding sites are located on the molecular surface at both ends of the dumbbell-shape molecule. The crystal of the NAG3 complex contains two independent molecules forming a protein-sugar 2:2 complex. One NAG3 molecule is sandwiched between two independent UDA-VI molecules and the other sugar molecule is also sandwiched by one UDA-VI molecule and symmetry-related another one. The sugar binding site of N-terminal domain consists of three subsites accommodating NAG3 while two NAG residues are bound to the C-terminal domain. In each sugar-binding site, three aromatic amino acid residues and one serine residue participate to the NAG3 binding. The sugar rings bound to two subsites are stacked to the side-chain groups of tryptophan or histidine and a tyrosine residue is in face-to-face contact with an acetylamino group, to which the hydroxyl group of a serine residue is hydrogen-bonded. The third subsite of the N-terminal domain binds a NAG moiety with hydrogen bonds. The results suggest that the triad of aromatic amino acid residues is intrinsic in sugar binding of hevein-like domains.     

Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids.

The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully processed Pra (N0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.     

GlyGly-CTERM and rhombosortase: a C-terminal protein processing signal in a many-to-one pairing with a rhomboid family intramembrane serine protease

The rhomboid family of serine proteases occurs in all domains of life. Its members contain at least six hydrophobic membrane-spanning helices, with an active site serine located deep within the hydrophobic interior of the plasma membrane. The model member GlpG from Escherichia coli is heavily studied through engineered mutant forms, varied model substrates, and multiple X-ray crystal studies, yet its relationship to endogenous substrates is not well understood. Here we describe an apparent membrane anchoring C-terminal homology domain that appears in numerous genera including Shewanella, Vibrio, Acinetobacter, and Ralstonia, but excluding Escherichia and Haemophilus. Individual genomes encode up to thirteen members, usually homologous to each other only in this C-terminal region. The domain's tripartite architecture consists of motif, transmembrane helix, and cluster of basic residues at the protein C-terminus, as also seen with the LPXTG recognition sequence for sortase A and the PEP-CTERM recognition sequence for exosortase. Partial Phylogenetic Profiling identifies a distinctive rhomboid-like protease subfamily almost perfectly co-distributed with this recognition sequence. This protease subfamily and its putative target domain are hereby renamed rhombosortase and GlyGly-CTERM, respectively. The protease and target are encoded by consecutive genes in most genomes with just a single target, but far apart otherwise. The signature motif of the Rhombo-CTERM domain, often SGGS, only partially resembles known cleavage sites of rhomboid protease family model substrates. Some protein families that have several members with C-terminal GlyGly-CTERM domains also have additional members with LPXTG or PEP-CTERM domains instead, suggesting there may be common themes to the post-translational processing of these proteins by three different membrane protein superfamilies.     

Hepatitis C virus-encoded NS2-3 protease: cleavage-site mutagenesis and requirements for bimolecular cleavage.

Cleavage at the 2/3 site of hepatitis C virus (HCV) is thought to be mediated by a virus-encoded protease composed of the region of the polyprotein encoding NS2 and the N-terminal one-third of NS3. This protease is distinct from the NS3 serine protease, which is responsible for downstream cleavages in the nonstructural region. Site-directed mutagenesis of residues surrounding the 2/3 cleavage site showed that cleavage is remarkably resistant to single-amino-acid substitutions from P5 to P3' (GWRLL decreases API). The only mutations which dramatically inhibited cleavage were the ones most likely to alter the conformation of the region, such as Pro substitutions at the P1 or P1' position, deletion of both amino acids at P1 and P1', or simultaneous substitution of multiple Ala residues. Cotransfection experiments were done to provide additional information on the polypeptide requirements for bimolecular cleavage. Polypeptides used in these experiments contained amino acid substitutions and/or deletions in NS2 and/or the N-terminal one-third of NS3. Polypeptides with defects in either NS2 or the N-terminal portion of NS3 but not both were cleaved when cotransfected with constructs expressing intact versions of the defective region. Cotransfection experiments also showed that certain defective NS2-3 constructs partially inhibited cleavage of wild-type polypeptides. Although these results show that inefficient cleavage can occur in a bimolecular reaction, they suggest that both molecules must contribute a functional subunit to allow formation of a protease which is capable of cleavage at the 2/3 site. This reaction may resemble the cis cleavage thought to occur at the 2/3 site during processing of the wild-type HCV polyprotein.     

Two Clip Domain Family of Serine Proteases and a Serine Protease Homolog from the Fall Webworm, Hyphantria cunea; cDNA Cloning and Characterization

Two types of serine proteases and a serine protease homologue cDNAs were isolated from Hyphantria cunea larvae induced immune response due to an injection of a microorganism through RT‐PCR and cDNA library screening, and their characteristics were examined. The isolated cDNAs are composed 2.1 kb, 2.2 kb, and 2.5 kb nucleotide each, which encoded 388, 390, 580 amino acid residues, and were designated as HcPE‐1, HcPE‐2 and HcPE‐3, respectively. They were revealed as serine proteases or a serine protease homologue with the clip domain through a database search. The deduced amino acid sequence comparison showed high homology of 72‐78% among them. Six Cys residues of the N‐terminal clip domain forming the disulfide bond, Cys residues of the catalytic domain, and Cys residues forming inter‐bridge between clip domain and catalytic domain were also well preserved. Three amino acid residues, His, Asp, and Ser, within the active site were perfectly conserved in HcPE‐2 and HcPE‐3, however, His was replaced with Gln¹⁷⁸ in HcPE‐1. The Arg residues (HcPE‐1, Arg¹³²; HcPE‐2, Arg¹³⁴; HcPE‐3, Arg³²⁵) known as the activation sites by proteolytic cleavage were preserved well in all three types of protein. In case of HcPE‐3, three continuous clip‐like domains existed in the N terminal. As the result of phylogenetic analysis, three clip domain family of protein from H. cunea make groups with arthropod proclotting enzyme precursor. Northern blot analysis showed all three genes were induced through an injection of Escherichia coli, but expression patterns were varied.     

Computational study of the putative active form of protein Z (PZa): sequence design and structural modeling

Although protein Z (PZ) has a domain arrangement similar to the essential coagulation proteins FVII, FIX, FX, and protein C, its serine protease (SP)-like domain is incomplete and does not exhibit proteolytic activity. We have generated a trial sequence of putative activated protein Z (PZa) by identifying amino acid mutations in the SP-like domain that might reasonably resurrect the serine protease catalytic activity of PZ. The structure of the activated form was then modeled based on the proposed sequence using homology modeling and solvent-equilibrated molecular dynamics simulations. In silico docking of inhibitors of FVIIa and FXa to the putative active site of equilibrated PZa, along with structural comparison with its homologous proteins, suggest that the designed PZa can possibly act as a serine protease.     

Carboxy-terminal cleavage of the human foamy virus Gag precursor molecule is an essential step in the viral life cycle.

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked.     

Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization

Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.