Kinetic Dissection of the Pre-existing Conformational Equilibrium in the Trypsin Fold

The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

Sørensen

Madsen

Svensson

et al. 2016

“…6) suggests that HABP2 G221E lacks the stabilization of the S1 pocket that normally results from N terminus insertion. As such, the activated form of HABP2 G221E may predominantly be inactive, presumably because of a shift of the equilibrium between closed (E*) and open (E) forms (23) or in the continuum between zymogen-and proteinase-like states (24).…”

Section: Hydrolysis Of S-2288mentioning

confidence: 99%

Role of glycine 221 in catalytic activity of hyaluronan-binding protein 2

Stavenuiter

Ebberink

Mertens

et al. 2017

Edited by Norma Allewell HABP2 (hyaluronan-binding protein 2) is a Ca2؉ -dependent serine protease with putative roles in blood coagulation and fibrinolysis. A G221E substitution, known as the Marburg I polymorphism, reportedly affects HABP2 function and has been associated with increased risk for cardiovascular disease. However, the importance of Gly-221 for HABP2 activity is unclear. Here, we used G221E, G221A, and G221S mutants to assess the role of Gly-221 in HABP2 catalysis. The G221E variant failed to activate the single-chain urokinase-type plasminogen activator, and the G221A and G221S variants displayed moderately reduced single-chain urokinase-type plasminogen activator activation. Activity toward the peptide substrate S-2288 was markedly decreased in all HABP2 variants, with G221E being the most defective and G221A being the least defective. In the absence of Ca 2؉ , S-2288 cleavage by wild-type HABP2 was Na ؉ -dependent, with K m decreasing from 3.0 to 0.6 mM upon titration from 0 to 0.3 M Na ؉ . In the presence of 5 mM Ca 2؉ , K m was further reduced to 0.05 mM, but without an appreciable contribution of Na ؉ . At physiological concentrations of Na ؉ and Ca 2؉ , the three HABP2 variants, and particularly G221E, displayed a major K m increase for S-2288. Chemical footprinting revealed that Ile-16 is significantly less protected from chemical modification in G221E than in wild-type HABP2, suggesting impaired insertion of the N terminus into the G221E protease domain, with a concomitant impact on catalytic activity. Homology modeling suggested that the Glu-221 side chain could sterically hinder insertion of the N terminus into the HABP2 protease domain, helping to explain the detrimental effects of Glu-221 substitution on HABP2 activity.

“…For the different zymogens, the pre-existing E 7 E* equilibrium is variably shifted to the E* form with equilibrium constants (K eq ) ranging from 10 8 (trypsinogen) (12) to 7 (tissue plasminogen activator) (13). For Pre2, a K eq of 10 has been recently determined (14). An indirect, albeit stringent, proof of the existence of the E 7 E* equilibrium in ProT is that protein binding to thrombin precursors can effectively stabilize the active E form, as demonstrated in the case of staphylocoagulase (SC) (15) and von Willebrand factor-binding protein (vWbp) (10), two proteins secreted from the virulent Gram-positive bacterium Staphylococcus aureus exploiting a molecular mimicry mechanism to orient their N-terminal Ile-1-Val-2 dipeptide into the Asp-524 cavity, thus leading to non-proteolytic activation of ProT.…”

mentioning

confidence: 99%

Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulant–anticoagulant equilibrium toward thrombosis

Pontarollo

Acquasaliente

Peterle

et al. 2017

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.Blood coagulation is a finely regulated physiological process that culminates with the factor Xa-mediated conversion of the prothrombin (ProT) 3 zymogen to the active ␣-thrombin (␣T) enzyme, which in turn is responsible for the generation of insoluble fibrin and activation of platelets via the GpIb␣-PAR1 pathway (1, 2). ProT (ϳ72 kDa) is a vitamin K-dependent glycoprotein produced in the liver and circulating at a relatively high plasma concentration (0.1 mg/ml) (3). The domain architecture of ProT includes a Gla domain (residues 1-46), a kringle-1 (residues 65-143) and a kringle-2 (residues 170 -248) domain, and a chymotrypsin-like protease domain (residues 285-579) connected by three intervening linker regions (Lnk-1, -2, and -3) (4). Isolated factor Xa (FXa) has low intrinsic ProTconverting activity, but when it is assembled in the presence of Ca 2ϩ with cofactor Va in the prothrombinase complex on the platelet surface, its ability to activate ProT is increased by about 5 orders of magnitude (5). FXa cleaves ProT in a concerted manner at two sites, i.e. Arg-271 and Arg-320, but the order of peptide bond cleavage is highly context-dependent. On the platelet surface, FXa first cleaves ProT at Arg-271 generating the inactive precursor prethrombin-2 (Pre2), which is attacked by FXa at Arg-320 to generate the active ␣T species, formed by the polypeptide chains Thr-272-Arg-320 and Ile-321-Glu-579 (6). ...