Von Willebrand factor-binding protein is a hysteretic conformational activator of prothrombin.
ABSTRACT: Von Willebrand factor-binding protein (VWbp), secreted by Staphylococcus aureus, displays secondary structural homology to the 3-helix bundle, D1 and D2 domains of staphylocoagulase (SC), a potent conformational activator of the blood coagulation zymogen, prothrombin (ProT). In contrast to the classical proteolytic activation mechanism of trypsinogen-like serine proteinase zymogens, insertion of the first 2 residues of SC into the NH(2)-terminal binding cleft on ProT (molecular sexuality) induces rapid conformational activation of the catalytic site. Based on plasma-clotting assays, the target zymogen for VWbp may be ProT, but this has not been verified, and the mechanism of ProT activation is unknown. We demonstrate that VWbp activates ProT conformationally in a mechanism requiring its Val(1)-Val(2) residues. By contrast to SC, full time-course kinetic studies of ProT activation by VWbp demonstrate that it activates ProT by a substrate-dependent, hysteretic kinetic mechanism. VWbp binds weakly to ProT (K(D) 2.5 microM) to form an inactive complex, which is activated through a slow conformational change by tripeptide chromogenic substrates and its specific physiological substrate, identified here as fibrinogen (Fbg). This mechanism increases the specificity of ProT activation by delaying it in a slow reversible process, with full activation requiring binding of Fbg through an exosite expressed on the activated ProT*.VWbp complex. The results suggest that this unique mechanism regulates pathological fibrin (Fbn) deposition to VWF-rich areas during S. aureus endocarditis.
Project description:Thrombin generation and fibrinogen (Fbg) clotting are the ultimate proteolytic reactions in the blood coagulation pathway. Staphylocoagulase (SC), a protein secreted by the human pathogen Staphylococcus aureus, activates prothrombin (ProT) without proteolysis. The SC.(pro)thrombin complex recognizes Fbg as a specific substrate, converting it directly into fibrin. The crystal structure of a fully active SC fragment containing residues 1-325 (SC-(1-325)) bound to human prethrombin 2 showed previously that SC inserts its Ile(1)-Val(2) N terminus into the Ile(16) pocket of prethrombin 2, inducing a functional active site in the cognate zymogen conformationally. Exosite I of alpha-thrombin, the Fbg recognition site, and proexosite I on ProT are blocked by domain 2 of SC-(1-325). In the present studies, active site-labeled fluorescent ProT analogs were used to quantitate Fbg binding to the SC-(1-325).ProT complex. Fbg binding and cleavage are mediated by expression of a new Fbg-binding exosite on the SC-(1-325).ProT complex, resulting in formation of an (SC-(1-325).ProT)(2).Fbg pentameric complex with a dissociation constant of 8-34 nm. In both crystal structures, the SC-(1-325).(pre)thrombin complexes form dimers, with both proteinases/zymogens facing each other over a large U-shaped cleft, through which the Fbg substrate could thread. On this basis, a molecular model of the pentameric (SC-(1-325).thrombin)(2).Fbg encounter complex was generated, which explains the coagulant properties and efficient Fbg conversion. The results provide new insight into the mechanism that mediates high affinity Fbg binding and cleavage as a substrate of SC.(pro)thrombin complexes, a process that is central to the molecular pathology of S. aureus endocarditis.
Project description:Staphylocoagulase (SC) is a protein secreted by the human pathogen, Staphylococcus aureus, that activates human prothrombin (ProT) by inducing a conformational change. SC-bound ProT efficiently clots fibrinogen, thus bypassing the physiological blood coagulation pathway. The crystal structure of a fully active SC fragment, SC-(1-325), bound to human prethrombin 2 showed that the SC-(1-325) N terminus inserts into the Ile(16) pocket of prethrombin 2, thereby inducing expression of a functional catalytic site in the cognate zymogen without peptide bond cleavage. As shown here, SC-(1-325) binds to bovine and human ProT with similar affinity but activates the bovine zymogen only very poorly. By contrast to the approximately 2-fold difference in chromogenic substrate kinetic constants between human thrombin and the SC-(1-325).human (pro)thrombin complexes, SC-(1-325).bovine ProT shows a 3,500-fold lower k(cat)/K(m) compared with free bovine thrombin, because of a 47-fold increase in K(m) and a 67-fold decrease in k(cat). The SC-(1-325).bovine ProT complex is approximately 5,800-fold less active compared with its human counterpart. Comparison of human and bovine fibrinogen as substrates of human and bovine thrombin and the SC-(1-325).(pro)thrombin complexes indicates that the species specificity of SC-(1-325) cofactor activity is determined primarily by differences in conformational activation of bound ProT. These results suggest that the catalytic site in the SC-(1-325).bovine ProT complex is incompletely formed. The current crystal structure of SC-(1-325).bovine thrombin reveals that SC would dock similarly to the bovine proenzyme, whereas the bovine (pro)thrombin-characteristic residues Arg(144) and Arg(145) would likely interfere with insertion of the SC N terminus, thus explaining the greatly reduced activation of bovine ProT.
Project description:Staphylocoagulase (SC) secreted by Staphylococcus aureus is a potent non-proteolytic activator of the blood coagulation zymogen prothrombin and the prototype of a newly established zymogen activator and adhesion protein (ZAAP) family. The conformationally activated SC.prothrombin complex specifically cleaves fibrinogen to fibrin, which propagates the growth of bacteria-fibrin-platelet vegetations in acute bacterial endocarditis. Our recent 2.2 A X-ray crystal structures of an active SC fragment [SC(1-325)] bound to the prothrombin zymogen catalytic domain, prethrombin 2, demonstrated that SC(1-325) represents a new type of non-proteolytic activator with a unique fold. The observed insertion of the SC(1-325) N-terminus into the 'Ile 16' cleft of prethrombin 2, which triggers the activating conformational change, provided the first unambiguous structural evidence for the 'molecular sexuality' mechanism of non-proteolytic zymogen activation. Based on the SC(1-325) fold, a new family of bifunctional zymogen activator and adhesion proteins was identified that possess N-terminal domains homologous to SC(1-325) and C-terminal domains that mediate adhesion to plasma or extracellular matrix proteins. Further investigation of the ZAAP family may lead to new insights into the mechanisms of bacterial factors that hijack zymogens of the human blood coagulation and fibrinolytic systems to promote and disseminate endocarditis and other infectious diseases.
Project description:Staphylococcus aureus is the leading cause of infective endocarditis (IE). While the role of S. aureus cell-wall associated protein clumping factor A (ClfA) in promoting IE has been already demonstrated, that of the secreted plasma-clotting factors staphylocoagulase (Coa) and von Willebrand factor-binding protein (vWbp) has not yet been elucidated. We investigated the role of Coa and vWbp in IE initiation in rats with catheter-induced aortic vegetations, using Lactococcus lactis expressing coa, vWbp, clfA or vWbp/clfA, and S. aureus Newman ?coa, ?vWbp, ?clfA or ?coa/?vWbp/?clfA mutants. vWbp-expression increased L. lactis valve infection compared to parent and coa-expressing strains (incidence: 62%, versus 0% and 13%, respectively; P < 0.01). Likewise, expression of clfA increased L. lactis infectivity (incidence: 80%), which was not further affected by co-expression of vWbp. In symmetry, deletion of the coa or vWbp genes in S. aureus did not decrease infectivity (incidence: 68 and 64%, respectively) whereas deletion of clfA did decrease valve infection (incidence: 45%; P = 0.03 versus parent), which was not further affected by the triple deletion ?coa/?vWbp/?clfA (incidence: 36%; P > 0.05 versus ?clfA mutant). Coa does not support the initial colonization of IE (in L. lactis) without other key virulence factors and vWbp contributes to initiation of IE (in L. lactis) but is marginal in the present of ClfA.
Project description:The two coagulases, von Willebrand factor binding protein (vWbp) and Coagulase (Coa), are critical virulence factors in several animal models of invasive Staphylococcus aureus (S. aureus) infections. These proteins are part of an intricate system of proteins that S. aureus uses to assemble a fibrinogen (Fg)/fibrin protective shield surrounding itself. This shield allows the microorganism to evade clearance by the host phagocytic cells. The coagulases can non-proteolytically activate the zymogen prothrombin to convert Fg to fibrin and promote the Fg/fibrin shield formation. The coagulases also bind directly to Fg and the interaction between Coa and Fg has been previously characterized in some detail. However, the mechanism(s) by which vWbp interacts with Fg remains unclear. Here, we show that vWbp and Coa have distinct interactions with Fg, despite being structurally similar. Coa binds with a significantly higher affinity to soluble Fg than to Fg coated on a plastic surface, whereas vWbp demonstrates no preference between the two forms of Fg. The two coagulases appear to target different sites on Fg, as they do not compete with each other in binding to Fg. Similar to Coa, both the N- and C-terminal halves of vWbp (vWbp-N, vWbp-C, respectively) harbor Fg-binding activities. The higher affinity Fg-binding activity resides in vWbp-N; whereas, the C-terminal region of Coa encompasses the major Fg-binding activity. Peptides constituting the previously identified Coa/Efb1 Fg-binding motif fail to inhibit vWbp-C from binding to Fg, indicating that vWbp-C lacks a functional homolog to this motif. Interestingly, the N-terminal prothrombin-binding domains of both coagulases recognize the Fg ?-chain, but they appear to interact with different sequence motifs in the host protein. Collectively, our data provide insight into the complex interactions between Fg and the S. aureus coagulases.
Project description:A flexible linker (Lnk2) composed of 26 amino acids connects kringle-1 to kringle-2 in the coagulation factor prothrombin. Recent studies point to Lnk2 as a key determinant of the structure and function of this zymogen. Using a combination of mutagenesis, structural biology, and single molecule spectroscopy, we show how Lnk2 influences activation and conformational plasticity of prothrombin. Scrambling the sequence of Lnk2 is inconsequential on activation, and so is extension by as many as 22 residues. On the other hand, below a critical length of 15 residues, the rate of prothrombin activation increases (10-fold) in the absence of cofactor Va and decreases (3-fold) in the presence of cofactor. Furthermore, activation by prothrombinase takes place without preference along the prethrombin-2 (cleavage at Arg(271) first) or meizothrombin (cleavage at Arg(320) first) pathways. Notably, these transitions in the rate and pathway of activation require the presence of phospholipids, pointing to an important physiological role for Lnk2 when prothrombin is anchored to the membrane. Two new crystal structures of prothrombin lacking 22 (ProT?146-167) or 14 (ProT?154-167) residues of Lnk2 document striking conformational rearrangements of domains located across this linker. FRET measurements of freely diffusing single molecules prove that these structural transitions are genuine properties of the zymogen in solution. These findings support a molecular model of prothrombin activation where Lnk2 presents the sites of cleavage at Arg(271) and Arg(320) to factor Xa in different orientations by pivoting the C-terminal kringle-2/protease domain pair on the N-terminal Gla domain/kringle-1 pair anchored to the membrane.
Project description:Prion diseases involve the conformational conversion of the cellular prion protein (PrP(C)) to its misfolded pathogenic form (PrP(Sc)). To better understand the structural mechanism of this conversion, we performed extensive all-atom, explicit-solvent molecular-dynamics simulations for three structures of the wild-type human PrP (huPrP) at different pH values and temperatures. Residue 129 is polymorphic, being either Met or Val. Two of the three structures have Met in position 129 and the other has Val. Lowering the pH or raising the temperature induced large conformational changes of the C-terminal globular domain and increased exposure of its hydrophobic core. In some simulations, HA and its preceding S1-HA loop underwent large displacements. The C-terminus of HB was unstable and sometimes partially unfolded. Two hydrophobic residues, Phe-198 and Met-134, frequently became exposed to solvent. These conformational changes became more dramatic at lower pH or higher temperature. Furthermore, Tyr-169 and the S2-HB loop, or the X-loop, were different in the starting structures but converged to common conformations in the simulations for the Met-129, but not the Val-129, protein. α-Strands and β-strands formed in the initially unstructured N-terminus. α-Strand propensity in the N-terminus was different between the Met-129 and Val129 proteins, but β-strand propensity was similar. This study reveals detailed structural and dynamic properties of huPrP, providing insight into the mechanism of the conversion of PrP(C) to PrP(Sc).
Project description:Dimeric effectors caspase 3 and caspase 7 are activated by initiator caspase processing. In this study, we report the crystal structures of effector caspase 6 (CASP6) zymogen and N-Acetyl-Val-Glu-Ile-Asp-al-inhibited CASP6. Both of these forms of CASP6 have a dimeric structure, and in CASP6 zymogen the intersubunit cleavage site (190)TEVD(193) is well structured and inserts into the active site. This positions residue Asp 193 to be easily attacked by the catalytic residue Cys 163. We demonstrate biochemically that intramolecular cleavage at Asp 193 is a prerequisite for CASP6 self-activation and that this activation mechanism is dependent on the length of the L2 loop. Our results indicate that CASP6 can be activated and regulated through intramolecular self-cleavage.
Project description:Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active ?-thrombin (?T). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor prethrombin-2 (Pre2), which is further attacked at Arg-320-Ile-321 to yield mature ?T. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nm to 2 ?m), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active ?Pre2, a nicked Pre2 derivative with a single cleaved Ala-470-Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470-Asn-471 is instrumental for the onset of catalysis in ?Pre2, which was, however, reduced about 100-200-fold compared with ?T. Of note, ?Pre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.
Project description:Thrombin, derived from zymogen prothrombin (ProT), is a serine protease involved in procoagulation, anticoagulation, and platelet activation. Thrombin's actions are regulated through anion-binding exosites I and II (ABE I and ABE II) that undergo maturation during activation. Mature ABEs can utilize exosite-based communication to fulfill thrombin functions. However, the conformational basis behind such long-range communication and the resultant ligand binding affinities are not well understood. Protease activated receptors (PARs), involved in platelet activation and aggregation, are known to target thrombin ABE I. Unexpectedly, PAR3 (44-56) can already bind to pro-ABE I of ProT. Nuclear magnetic resonance (NMR) ligand-enzyme titrations were used to characterize how individual PAR1 (49-62) residues interact with pro-ABE I and mature ABE I. 1D proton line broadening studies demonstrated that binding affinities for native PAR1P (49-62, P54) and for the weak binding variant PAR1G (49-62, P54G) increased as ProT was converted to mature thrombin. 1H,15N-HSQC titrations revealed that PAR1G residues K51, E53, F55, D58, and E60 exhibited less affinity to pro-ABE I than comparable residues in PAR3G (44-56, P51G). Individual PAR1G residues then displayed tighter binding upon exosite maturation. Long-range communication between thrombin exosites was examined by saturating ABE II with phosphorylated GpIb? (269-282, 3Yp) and monitoring the binding of PAR1 and PAR3 peptides to ABE I. Individual PAR residues exhibited increased affinities in this dual-ligand environment supporting the presence of interexosite allostery. Exosite maturation and beneficial long-range allostery are proposed to help stabilize an ABE I conformation that can effectively bind PAR ligands.