Engineering Thrombin for Selective Specificity toward Protein C and PAR1

Abstract

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...

Autoactivation of Thrombin Precursors

“…Production of the mutant EDGE with the additional S195A substitution as a control and for crystallization failed repeatedly due to lack of refolding. Prethrombin-1 wild-type and mutants EDE and EDES were expressed in baby hamster kidney cells as described (28). Benzamidine was added after collecting the cell supernatant at a final concentration of 10 mM to prevent proteolysis.…”

“…Benzamidine was added after collecting the cell supernatant at a final concentration of 10 mM to prevent proteolysis. Gla domainless prothrombin wild-type and mutants EDE and EDES were expressed in baby hamster kidney cells (28) after introduction of an ApaI restriction site in the human prothrombin cDNA. Homogeneity and chemical identity of final preparations were verified by SDS-PAGE and by reverse phase HPLC mass spectrometry analysis, giving a purity of Ͼ98%.…”

“…Samples were processed by electrophoresis using 4 -12% NuPAGE gels and MES running buffer, stained with Coomassie Brilliant Blue R-250, and analyzed by quantitative densitometry. The activity of the autoactivated constructs was tested against the chromogenic substrate H-D-Phe-Pro-Arg-p-nitroanilide and physiological substrates fibrinogen, PAR1, and protein C in the presence of 100 nM thrombomodulin and 5 mM CaCl 2 under experimental conditions of 5 mM Tris, pH 7.4, 0.1% PEG 8000, 145 mM NaCl, 37°C, as detailed elsewhere (28). Proteolytically degraded ␤-thrombin and ␥-thrombin were removed by hydrophobic interaction chromatography on a TSK-Gel Phenyl 5PW column (75 ϫ 7.5 mm, 10 m, Supleco).…”

Structural basis of thrombin–protease‐activated receptor interactions

“…Exosites are spatially separated from the active site region, but linked to it by allosteric communication (23)(24)(25). Exosite I occupies a position homologous to the Ca 21 binding loop of trypsin and chymotrypsin, 20-25 Å away from the active site moiety and is a binding epitope for fibrinogen (26,27), thrombomodulin (27)(28)(29), and the thrombin receptors PAR1 (16,26,30,31) and PAR3 (17,32). The potent natural inhibitor hirudin also targets exosite I via its extended, acidic C-terminal domain (33).…”

Switching cation-binding loops paves the way for redesigning allosteric activation

“…Some mutants that have already been designed to convert thrombin into an effective anticoagulant include E217K, W215A/E217A (WE), W215E, and Δ146-149e (16)(17)(18). The most extensive in vivo testing has been done with the WE mutant.…”