Important Role of the Cys-191–Cys-220 Disulfide Bond in Thrombin Function and Allostery

Self Cite

Thrombin elicits functional responses critical to blood homeostasis by interacting with diverse physiological substrates. Ala-scanning mutagenesis of 97 residues covering 53% of the solvent accessible surface area of the enzyme identifies Trp 215 as the single most important determinant of thrombin specificity. Saturation mutagenesis of Trp 215 produces constructs featuring k cat /K m values for the hydrolysis of fibrinogen, protease-activated receptor PAR1, and protein C that span five orders of magnitude. Importantly, the effect of Trp 215 replacement is context dependent. Mutant W215E is 10-fold more specific for protein C than fibrinogen and PAR1, which represents a striking shift in specificity relative to wild-type that is 100-fold more specific for fibrinogen and PAR1 than protein C. However, when the W215E mutation is combined with deletion of nine residues in the autolysis loop, which by itself shifts the specificity of the enzyme from fibrinogen and PAR1 to protein C, the resulting construct features significant activity only toward PAR1. These findings demonstrate that thrombin can be re-engineered for selective specificity toward protein C and PAR1. Mutations of Trp 215 provide important reagents for dissecting the multiple functional roles of thrombin in the blood and for clinical applications.Engineering protease specificity remains an issue of considerable interest and importance (1). Within the same fold, trypsins prefer Arg/Lys side chains at the P1 position (2) of substrate but chymotrypsins prefer Phe/Tyr/Trp residues (3-6). Primary specificity, defined by the nature of the P1 residue, is not the sole determinant of protease function. Many trypsin-like proteases show substrate preference that extends beyond the P1 position of substrate and is dictated by interactions with other structural domains not in contact with the primary specificity pocket. Indeed, inspection of consensus sequences recognized by thrombin for its three primary physiological targets, i.e. the procoagulant substrate fibrinogen, the prothrombotic protease-activated receptor 1 (PAR1) 2 and the anticoagulant substrate protein C, reveals an Arg residue at the P1 position in all cases (7). Hence, selection among these targets must depend on interactions beyond the primary specificity pocket of the enzyme. A paradigm widely accepted in the field is that macromolecular specificity in thrombin and related clotting proteases is achieved and controlled by interaction with "exosites," i.e. domains widely separated from the active site region that kinetically control docking of substrate in ways that restrict choices by the enzyme (8, 9). However, abundant mutagenesis data show that residues within the active site play roles that far exceed in importance those of exosites and, in fact, affect specificity and the choice of which substrate can be cleaved by the enzyme in ways that are unmatched by other domains (10 -13). Consistent with these findings, the present study demonstrates that residue 215 within the thrombin active site is th...

Section: Methodssupporting

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Engineering Thrombin for Selective Specificity toward Protein C and PAR1

Marino

Pelc

Vogt

et al. 2010

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“…Mutations in thrombin that destabilize the proteinase would adversely affect Na ϩ binding and similarly affect function, appearing as anticoagulant thrombins (11,12). This likely accounts for the diverse range of mutations in thrombin, including removal of several residues in the autolysis loop or a highly conserved disulfide bond, that have been shown to yield varyingly anticoagulant-specific or E* proteinase forms (11,42). In line with this reasoning, distorted structures reported for various thrombin mutants are rectified to proteinase-like structures following reaction with FPRck or complexation with ABE1 ligands (32,34).…”

Section: Discussionmentioning

confidence: 99%

Ligand Binding Shuttles Thrombin along a Continuum of Zymogen- and Proteinase-like States

Kamath

Huntington

Krishnaswamy

2010

The critical and multiple roles of thrombin in blood coagulation are regulated by ligands and cofactors. Zymogen activation imparts proteolytic activity to thrombin and also affects the binding of ligands to its two principal exosites. We have used the activation peptide fragment 1.2 (F12), a ligand for anion-binding exosite 2, to probe the zymogenicity of thrombin by isothermal titration calorimetry. We show that F12 binding is sensitive to subtle aspects of proteinase formation beyond simply reporting on zymogen cleavage. Large thermodynamic differences in F12 binding distinguish between a series of thrombin species poised along the transition of zymogen to proteinase. Thrombin is a pivotal serine proteinase product of the blood coagulation cascade. In addition to catalyzing fibrinogen cleavage and platelet activation to form the blood clot, it also plays essential regulatory roles (1, 2). It catalyzes proteolytic activation steps that amplify flux toward thrombin formation following initiation of the coagulation cascade. Thrombin also functions as a negative regulator by activating protein C in the anticoagulant pathway (3). These opposing roles of thrombin in coagulation derive from its ability to act with specificity on a range of protein substrates, with differing sequences flanking the cleavage sites, in reactions that are regulated by cofactors and two anion-binding exosites (ABE1 and ABE2) 2 found on opposite faces of the proteinase (4, 5). Allosteric control, arising from binding of ligands to different sites, is considered to underlie the regulation of thrombin specificity and its dual role as procoagulant and anticoagulant proteinase (4, 5). Thrombin and the other serine proteinases of blood coagulation contain catalytic domains bearing the chymotrypsin fold (6). In this family, the zymogen precursor is converted to proteinase by cleavage following Arg 15 (7). 3 The nascent N terminus inserts in a sequence-specific manner into an N-terminal binding pocket to form a salt bridge with Asp 194 (7). Salt bridge formation is associated with changes in activation domains, optimization of the primary specificity pocket to facilitate substrate binding, and a flip in the Gly 193 amide bond to form the oxyanion hole (7). These transitions imbue the product with proteolytic activity.Early studies with chymotrypsin suggested the persistence of zymogen-like species at near-neutral pH, related to the partial deprotonation of the new N terminus responsible for salt bridge formation (8). It is nevertheless implicitly assumed that irreversible cleavage at Arg 15 and the concerted transitions that ensue highly favor proteinase formation. This is despite the likely presence of intervening equilibria between substates that lie on the pathway for conversion of zymogen to fully stabilized proteinase. A notable exception is factor VIIa which possesses poor catalytic activity and has been proposed to be zymogenlike when free and stabilized in the proteinase state when bound to tissue factor (9). Na ϩ is considered an essenti...

“…In the rare blood coagulation disorder known as Hageman trait, Cys 571 is mutated to a serine in factor XII, which ablates the disulfide bond and inactivates the enzyme (45). Elimination of the disulfide bond in the blood coagulation protease thrombin (Cys 540 -Cys 571 in thrombin) results in a ϳ100-fold loss of activity toward peptide substrates (46). The disulfide bond in trypsin (Cys 173 -Cys 197 in trypsin) is also important for activity, although there are conflicting reports on the nature of the change in activity upon elimination of the bond.…”

Section: Conditionsmentioning

confidence: 99%

Allosteric Control of βII-Tryptase by a Redox Active Disulfide Bond

Cook

McNeil

Hogg

2013

Background:The S1A serine proteases function in many biological processes and contain a conserved disulfide bond near the active site.Results: This disulfide in the S1A mast cell protease, ␤II-tryptase, exists in oxidized and reduced states in the enzyme, which influences the specificity and efficiency of the enzyme. Conclusion: ␤II-Tryptase is regulated by an allosteric disulfide bond. Significance: Other S1A proteases are likely similarly regulated.