Characterization of a thrombomodulin binding site on protein C and its comparison to an activated protein C binding site for factor Va

Activation of the anticoagulant human plasma serine protease zymogen, protein C, by a complex of thrombin and the membrane protein, thrombomodulin, generates activated protein C, a physiologic anti-thrombotic, anti-inflammatory and anti-apoptotic agent. Alanine-scanning site-directed mutagenesis of residues in five surface loops of an extensive basic surface on protein C was used to identify residues that play essential roles in its activation by the thrombin-thrombomodulin complex. Twenty-three residues in the protein C protease domain were mutated to alanine, singly, in pairs or in triple mutation combinations, and mutants were characterized for their effectiveness as substrates of the thrombin-thrombomodulin complex. Three protein C residues, K192, R229, and R230, in two loops, were identified that provided major contributions to interactions with thrombin-thrombomodulin, while six residues, S190, K191, K217, K218, W231, and R312, in four loops, appeared to provide minor contributions. These protein C residues delineated a positively charged area on the molecule's surface that largely overlapped the previously characterized factor Va binding site on activated protein C. Thus, the extensive basic surface of protein C and activated protein C provides distinctly different, though significantly overlapping, binding sites for recognition by thrombin-thrombomodulin and factor Va.     

Nonenzymatic anticoagulant activity of the mutant serine protease Ser360Ala-activated protein C mediated by factor Va.

The human plasma serine protease, activated protein C (APC), primarily exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. A recombinant active site Ser 360 to Ala mutation of protein C was prepared, and the mutant protein was expressed in human 293 kidney cells and purified. The activation peptide of the mutant protein C zymogen was cleaved by a snake venom activator, Protac C, but the "activated" S360A APC did not have amidolytic activity. However, it did exhibit significant anticoagulant activity both in clotting assays and in a purified protein assay system that measured prothrombinase activity. The S360A APC was compared to plasma-derived and wild-type recombinant APC. The anticoagulant activity of the mutant, but not native APC, was resistant to diisopropyl fluorophosphate, whereas all APCs were inhibited by monoclonal antibodies against APC. In contrast to native APC, S360A APC was not inactivated by serine protease inhibitors in plasma and did not bind to the highly reactive mutant protease inhibitor M358R alpha 1 antitrypsin. Since plasma serpins provide the major mechanism for inactivating APC in vivo, this suggests that S360A APC would have a long half-life in vivo, with potential therapeutic advantages. S360A APC rapidly inhibited factor Va in a nonenzymatic manner since it apparently did not proteolyze factor Va. These data suggest that native APC may exhibit rapid nonenzymatic anticoagulant activity followed by enzymatic irreversible proteolysis of factor Va. The results of clotting assays and prothrombinase assays showed that S360A APC could not inhibit the variant Gln 506-FVa compared with normal Arg 506-FVa, suggesting that the active site of S360A APC binds to FVa at or near Arg 506.     

Charge reversal at the P3' position in protein C optimally enhances thrombin affinity and activation rate.

We have examined the properties of several human protein C (HPC) derivatives with substitutions for acidic residues near the thrombin cleavage site, including changing the P3' Asp to Asn (D172N), Gly (D172G), Ala (D172A), or Lys (D172K). The rate of thrombin-catalyzed activation of D172N, D172G, and D172A was increased 4-9-fold compared to wild-type HPC, primarily due to a reduction in the inhibitory effect of calcium and a resulting increase in affinity for free alpha-thrombin. There was no significant increase in activation rate or affinity with these 3 derivatives in the absence of calcium, confirming that P3' Asp affects calcium dependency in the native protein C molecule. With charge reversal at P3' (D172K), there was a 30-fold increase in activation rate in the presence of calcium, but unlike the other derivatives, there was a substantial effect (5-fold) on the activation rate and affinity for free alpha-thrombin in the absence of calcium. Thus, protein C affinity for thrombin appears to be influenced by a combination of calcium-dependent and -independent effects of the acidic P3' residue.     

Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation,proteolytic activity,and cell surface localization

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.     

Structure and evolution of the human genes encoding protein C and coagulation factor IX

Human protein C is a vitamin K-dependent plasma protein that serves as a feedback down-regulator of the coagulation cascade by specifically degrading the protein cofactors VIIIa and Va. The protein C precursor consists of the following domains: leader peptide, "gla" region, two epidermal growth factor segments, and the activation peptide/serine protease. Comparison of amino acid sequences reveals that protein C and factor IX are homologous. A comparison of the genes for protein C and factor IX shows that all seven of the introns within the protein coding regions are in identical positions and correspond to protein structure-function domain boundries. However, the base compositions of the two genes (coding and noncoding regions) are remarkably different: approximately 60% guanine + cytosine (G + C) for protein C versus approximately 40% G + C for factor IX. One possible explanation for this phenomenon is that the factor IX gene (located on the X chromosome) has undergone extensive deoxycytosine methylation and subsequent spontaneous deamination mutagenesis, resulting in a net C to thymine (and G to adenine) transition. This would suggest that the protein C gene may represent a more primitive form of the gene duplication precursor.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

Presence versus absence of hydrogen bond donor Tyr-39 influences interactions of cationic trypsin and mesotrypsin with protein protease inhibitors

Mesotrypsin displays unusual resistance to inhibition by polypeptide trypsin inhibitors and cleaves some such inhibitors as substrates, despite a high degree of conservation with other mammalian trypsins. Substitution of Arg for the generally conserved Gly-193 has been implicated as a critical determinant of the unusual behavior of mesotrypsin toward protein protease inhibitors. Another relatively conserved residue near the trypsin active site, Tyr-39, is substituted by Ser-39 in mesotrypsin. Tyr-39, but not Ser-39, forms a hydrogen bond with the main chain amide nitrogen of the P₄′ residue of a bound protease inhibitor. To investigate the role of the Tyr-39 H-bond in trypsin-inhibitor interactions, we reciprocally mutated position 39 in mesotrypsin and human cationic trypsin to Tyr-39 and Ser-39, respectively. We assessed inhibition constants and cleavage rates of canonical protease inhibitors bovine pancreatic trypsin inhibitor (BPTI) and the amyloid precursor protein Kunitz protease inhibitor domain by mesotrypsin and cationic trypsin variants, finding that the presence of Ser-39 relative to Tyr-39 results in a 4- to 13-fold poorer binding affinity and a 2- to 18-fold increase in cleavage rate. We also report the crystal structure of the mesotrypsin-S39Y•BPTI complex, in which we observe an H-bond between Tyr-39 OH and BPTI Ile-19 N. Our results indicate that the presence of Ser-39 in mesotrypsin, and corresponding absence of a single H-bond to the inhibitor backbone, makes a small but significant functional contribution to the resistance of mesotrypsin to inhibition and the ability of mesotrypsin to proteolyze inhibitors.     

重组人丝氨酸蛋白酶抑制因子Hespintor Kazal结构域的原核表达、纯化及活性鉴定

Hespintor是应用抑制消减杂交技术 (SSH) 从肝母细胞瘤细胞系HepG2中筛选得到的一未知功能蛋白,序列分析表明该蛋白属于Kazal型丝氨酸蛋白酶抑制因子 (Serine proteinase inhibitor, Serpin) 家族中的一个分泌型新成员,具有与食管癌相关基因2 (Esophageal cancer related gene 2, ECRG2) 高度同源的Serpin基本结构。为了进一步阐明Hespintor的生物学功能,必须得到纯化的Hespintor蛋白。先将Hespintor Kazal结构域编码序列亚克隆至原核表达载体pET-40b(+),转化至Rosetta (DE3) 表达宿主菌中。经 0.25 mmol/L IPTG,30 ℃诱导5 h获得了分子量约为42 kDa的Hespintor-Kazal重组融合蛋白的优化表达,Western blotting证实了重组蛋白的特异性。Hespintor-Kazal重组融合蛋白以包涵体形式在宿主菌中表达,利用金属螯合亲和层析和阴离子交换层析柱对重组蛋白进行两步纯化。初步的活性鉴定表明,纯化的Hespintor-Kazal重组融合蛋白能特异性抑制胰蛋白酶的水解活性,提示Hespintor具有作为一种新型抗肿瘤药物的潜在开发价值。     

Interactions of the C2 domain of human factor V with a model membrane

Activated coagulation Factor V is an important cofactor of the coagulation cascade that catalyzes the formation of the prothrombinase complex on the surface of membranes rich in phosphatidyl-L-serine (PS). Here we report molecular dynamics simulations of the two crystallographic structures (the open and closed conformations) of domain C2 of coagulation Factor V (FaVC2). The calculations were performed in water (1.5 ns for each conformation) and in the presence of a neutral phospholipid bilayer model (POPE; 10 ns for each conformation) in order to describe the dynamics of the free (plasma circulating) and membrane bound forms of FaVC2. Water simulations confirmed the hypothesis that the plasma circulating form is in the closed conformation. In contrast, the membrane simulations showed that both conformations are energetically compatible with membrane binding. We have investigated the mechanism, the dynamics, and the energetics of the binding process. Our data are consistent with published estimates of the immersion depth of the aromatic residues (W26 and W27), and with mutagenesis studies involving specific residues located on the spikes at the bottom of the FaVC2 structure. Electrostatic interactions between the phospholipid head groups and hydrophilic residues at the bottom of the structure play a key role in the binding process by creating a large number of hydrogen bonds that anchor the protein to the membrane. The simulations identified a stable phospholipid binding pocket reminiscent of a previously suggested PS interaction site. Our structural data could contribute to the design of potential inhibitors able to disrupt membrane association.     

Three-dimensional structural model of the serine receptor ligand-binding domain.

Computer-based homology modeling techniques were used to construct a three-dimensional model of the Escherichia coli serine receptor ligand-binding domain based on the crystal structure of the Salmonella typhimurium aspartate receptor and the sequence homology between the two receptors. Residues that have been found in mutagenesis studies to be necessary for serine binding are located in a proposed serine-binding site. Several other mutations that affect swimming behavior require relatively small shifts in alpha-carbon positions in the model to give a minimized structure, suggesting that small changes in receptor conformation can affect the signaling state of the receptor.     

Predicted three-dimensional structure of the protease inhibitor domain of the Alzheimer's disease beta-amyloid precursor

Alzheimer's disease is characterized by the deposition of amyloid beta-protein as plaques and tangles in the brains of its victims. The amyloid precursor can be expressed with or without the inclusion of a protease inhibitor domain, the potential role of which in amyloidogenesis has prompted the generation of a model of its three-dimensional structure based on the known structure of a related inhibitor. The model structure predicts that the mutated residues are almost entirely on the surface of the inhibitor domain, while conserved residues constitute the hydrophobic core. In addition, several pairs of structurally complementary, or concerted, mutations are seen. These structural features provide strong evidence for the validity of the modeled structure, and it is suggested that the presence of complementary mutations may be used as a criterion for evaluating protein structures built by homology, in addition to the (spatial) location of the mutations. The terminal residues delimiting the domain are among those furthest from the protease binding site and are in close proximity to one another, thus suggesting the ability of the domain to function as a structural cassette within the context of a larger protein. The electrostatic potentials of the inhibitor and of the related bovine pancreatic trypsin inhibitor reveal how two inhibitors with very different net charges can bind with approximately the same binding constant to trypsin and suggest a mutation of trypsin that might selectively enhance the binding of the amyloid inhibitor domain. The model provides a structural basis for understanding the functional roles of residues in the domain and for designing simpler molecules to test as pharmacologic agents for intervention in Alzheimer's disease.     

Synthesis of protein kinase Cδ C1b domain by native chemical ligation methodology and characterization of its folding and ligand binding

The C1b domain of protein kinase Cδ (PKCδ), a potent receptor for ligands such as diacylglycerol and phorbol esters, was synthesized by utilizing native chemical ligation. With this synthetic strategy, the domain was efficiently constructed and shown to have high affinity ligand binding and correct folding. The C1b domain has been utilized for the development of novel ligands for the control of phosphorylation by PKC family members. This strategy will pave the way for the efficient construction of C1b domains modified with fluorescent dyes, biotin, etc. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Exploration of protein–protein interaction effects on α-2-macroglobulin in an inhibition of serine protease through gene expression and molecular simulations studies

In Prophenoloxidase (ProPO) cascade, two targets namely serine protease and α-2-macroglobulin are key regulators involved in the defense system of crustaceans. In biological systems, routine role of cell systems requires the understanding in protein–protein interactions through experimental and theoretical concepts, which might yield useful insights into the cellular responses. Response of cells to regulating the immune system is governed by the interactions-involved biomolecular simulations. Unfortunately, studies on the inhibitors (SP and α-2M) that negatively regulate the proPO system or melanization in penaeid shrimp are not yet available. In order to understand how these interactions change the proPO mechanism in Indian white shrimp Fenneropenaeus indicus was determined. In F. indicus, innate immune system is in a sensitive balance of intricate interactions; elucidating these interactions by the integration of in silico and in vitro has great potential. We have determined the expression of both the SP and α-2M enzymes in regulatory mechanism, which are analyzed through qRT-PCR, protein–protein docking, and simulation studies. From this work, we propose a novel approach for studying an organism at the systems level by integrating genome-wide computational analysis and the gene expression data.     

Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101

A novel haloalkaliphilic, thermostable serine protease was purified from the extreme halophilic archaeon, Halogeometricum borinquense strain TSS101. The protease was isolated from a stationary phase culture, purified 116-fold with 18% yield and characterized biochemically. The molecular mass of the purified enzyme was estimated to be 86 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10.0 in 20% NaCl. The enzyme had high activity over the pH range from 6.0 to 10.0. Enzymatic activity was strongly inhibited by 1 mM phenyl methylsulfonyl fluoride, but activity was increased 59% by 0.1% cetyltrimethylammonium bromide. The enzyme exhibited relatively high thermal stability, retaining 80% of its activity after 1 h at 90 degrees C. Thermostability increased in the presence of Ca2+. The stability of the enzyme was maintained in 10% sucrose and in the absence of NaCl.     

Reconciling the lock-and-key and dynamic views of canonical serine protease inhibitor action

The efficiency of canonical serine protease inhibitors is conventionally attributed to the rigidity of their protease binding loop with no conformational change upon enzyme binding, yielding an example of the lock-and-key model for biomolecular interactions. However, solution-state structural studies revealed considerable flexibility in their protease binding loop. We resolve this apparent contradiction by showing that enzyme binding of small, 35-residue inhibitors is actually a dynamic conformer selection process on the nanosecond-timescale. Thus, fast timescale dynamics enables the association rate to be solely diffusion-controlled just like in the rigid-body model.     

Scanning mutagenesis studies reveal multiple distinct regions within the human protein kinase C alpha regulatory domain important for phorbol ester-dependent activation of the enzyme

While phorbol ester-binding sites within protein kinase C alpha (PKCalpha) have been identified and characterized utilizing fragments of the enzyme, it remains unclear whether additional regions within the enzyme may play an important role in its ability to be activated by phorbol ester. To examine this hypothesis, we generated 20 glutathione-S-transferase-tagged, V1-deficient, human PKCalpha holoenzyme constructs in which tandem six or 12 amino acid residue stretches along the full regulatory domain were changed to alanine residues. Each protein was assessed for its ability to bind phorbol ester and to induce growth repression when its catalytic activity was activated by phorbol ester upon expression in yeast cells. Mutagenesis of residues 99-158 potently reduced phorbol binding, consistent with previously published findings on the importance of the C1b region in phorbol binding. In addition, we identified a number of regions within the PKC regulatory domain that, when mutagenized, blocked the activation of PKC-mediated growth repression by phorbol ester while actually enhancing phorbol ester binding in vitro (residues 33-62, and 75-86). This study thus helps distinguish regions important for phorbol binding from regions important for the ability of phorbol ester to activate the enzyme. Our findings also suggest that multiple regions within C2 are necessary for full activation of the enzyme by phorbol ester, in particular residues 231-254. Finally, three regions, when mutagenized, completely, blocked catalytic domain activity in vivo (residues 33-62, 75-86, and 123-146), underscoring the important role of regulatory domain sequences in influencing catalytic domain function, even in the absence of the V1 region containing the pseudosubstrate sequence. This is the first tandem mutagenesis study for PKC that assesses the importance of regions for both phorbol binding and for phorbol-dependent activation in the context of the entire holoenzyme.     

The C2 domain calcium-binding motif: structural and functional diversity.

The C2 domain is a Ca(2+)-binding motif of approximately 130 residues in length originally identified in the Ca(2+)-dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin 1 and phosphoinositide-specific phospholipase C-delta 1, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation.     

The effect of calcium (II) on the binding of anticoagulation factor I with activated coagulation factor X

Anticoagulation factor I (ACF I) from the venom of Agkistrodon acutus forms a 1:1 complex with activated coagulation factor X (FXa) in a Ca²⁺-dependent fashion and thereby prolongs the clotting time. In the present study, the dependence of the binding of ACF I with FXa on the concentration of Ca²⁺ ions was quantitatively analyzed by HPLC, and the result showed that the maximal binding of ACF I to FXa occurred at concentration of Ca²⁺ ions of about 1 mM. The binding of Ca²⁺ ions to ACF I was investigated by equilibrium dialysis and two Ca²⁺-binding sites with different affinities were identified. At pH 7.6, the apparent association constants K₁ and K₂ for these two sites were (1.8 ± 0.5) × 10⁵ and (2.7 ± 0.6) × 10⁴ M^–1 (mean ± SE, n = 4), respectively. It was evident from the observation of Ca²⁺-induced changes in the intrinsic fluorescence of ACF I that ACF I underwent a conformational change upon binding of Ca²⁺ ions. The occupation of both Ca²⁺-binding sites in ACF I required a concentration of Ca²⁺ ions of about 1 mM, which is equal to the effective concentration of Ca²⁺ ions required both for maximal binding of ACF I to FXa and for the maximal enhancement of emission fluorescence of ACF I. It could be deduced from these results that the occupation of both Ca²⁺-binding sites in ACF I with Ca²⁺ ions and subsequent conformational rearrangement might be essential for the binding of ACF I to FXa.     

Activation of blood coagulation factor VIIa with cleaved tissue factor extracellular domain and crystallization of the active complex

Exposure of blood to tissue factor leads to the formation of a high affinity tissue factor/factor VIIa complex which initiates blood coagulation. As a first step toward obtaining structural information of this enzyme system, a complex of active-site inhibited factor VIIa (F.VIIai) and soluble tissue factor (sTF) was prepared for crystallization. Crystals were obtained, but only after long incubation times. Analysis by SDS-PAGE and mass spectrometry indicated the presence of sTF fragments similar to those formed by proteolytic digestion with subtilisin (Konigsberg, W., Nemerson, Y., Fang, C., Lin, T.-C. Thromb. Haemost. 69:1171, 1993). To test the hypothesis that limited proteolysis of sTF facilitated the crystallization of the complex, sTF fragments were generated by subtilisin digestion and purified. Analysis by tandem mass spectrometry showed the presence of nonoverlapping N- and C-terminal sTF fragments encompassing more than 90% of the tissue factor extracellular domain. Enzymatic assays and binding studies demonstrated that an equimolar mixture of N- and C-terminal fragments bound to factor VIIa and fully restored cofactor activity. A complex of F.VIIai and sTF fragments was prepared for crystallization. Crystals were obtained using microseeding techniques. The best crystals had maximum dimensions of 0.12 × 0.12 × 0.6 mm and showed diffraction to a resolution of 3 Å. © 1995 Wiley-Liss, Inc.     

Mutations at KFRDI and VGK domains of enterovirus 71 3C protease affect its RNA binding and proteolytic activities

The 3C proteases (3C^pro) of enterovirus 71 (EV71) is a good molecular target for drug discovery. Notably, this protease was found to possess RNA-binding activity. The regions responsible for RNA binding were classified as KFRDI (positions 82–86) and VGK (positions 154–156) in 3C^pro by mutagenesis study. Although the RNA-binding regions are structurally distinct from the catalytic site of EV71 3C^pro, mutations in the RNA-binding regions influenced 3C^pro proteolytic activity. In contrast, mutations at the catalytic site had almost no influence on RNA binding ability. We identified certain mutations within 3C^pro which abrogated both the RNA-binding activity of the expressed, recombinant, protease and the ability to rescue virus from an infectious full-length clone of EV71 (pEV71). Interestingly, mutation at position 84 from Arg(R) to Lys(K) was found to retain good RNA binding and proteolytic activity for the recombinant 3C^pro; however, no virus could be rescued when pEV71 with the R84K mutation was introduced into the infectious copy. Together, these results may provide useful information for using 3C^pro as the molecular target to develop anti-EV71 agents.The second and the third authors contributed equally to this work.