Abstract
Staphylocoagulase (SC) is a potent nonproteolytic prothrombin (ProT) activator and the prototype of a newly established zymogen activator and adhesion protein family. The staphylocoagulase fragment containing residues 1-325 (SC-(1-325)) represents a new type of nonproteolytic activator with a unique fold consisting of two three-helix bundle domains. The N-terminal, domain 1 of SC (D1, residues 1-146) interacts with the 148 loop of thrombin and prethrombin 2 and the south rim of the catalytic site, whereas domain 2 of SC (D2, residues 147-325) occupies (pro)exosite I, the fibrinogen (Fbg) recognition exosite. Reversible conformational activation of ProT by SC-(1-325) was used to create novel analogs of ProT covalently labeled at the catalytic site with fluorescence probes. Analogs selected from screening 10 such derivatives were used to characterize quantitatively equilibrium binding of SC-(1-325) to ProT, competitive binding with native ProT, and SC domain interactions. The results support the conclusion that SC-(1-325) binds to a single site on fluorescein-labeled and native ProT with indistinguishable dissociation constants of 17-72 pM. The results obtained for isolated SC domains indicate that D2 binds ProT with approximately 130-fold greater affinity than D1, yet D1 binding accounts for the majority of the fluorescence enhancement that accompanies SC-(1-325) binding. The SC-(1-325).(pro)thrombin complexes and free thrombin showed little difference in substrate specificity for tripeptide substrates or with their natural substrate, Fbg. Lack of a significant effect of blockage of (pro)exosite I of (pro)thrombin by SC-(1-325) on Fbg cleavage indicates that a new Fbg substrate recognition exosite is expressed on the SC-(1-325).(pro)thrombin complexes. Our results provide new insight into the mechanism that mediates zymogen activation by this prototypical bacterial activator.
Highlights
The recently determined crystal structure of a functional N-terminal fragment of SC (SC-(1–325)) bound to the catalytic domain of ProT, prethrombin 2 (Pre 2) (1) provided the first structural proof of the “molecular sexuality” mechanism of zymogen activation (7)
Our results demonstrate that SC-(1–325) binds to active site-labeled ProT analogs and native ProT with a 1:1 stoichiometry and extremely high affinity (KD ϭ 17–72 pM)
SC-(1– 325)1⁄7ProT complexes formed with native ProT were cleaved slowly at both thrombin-sensitive sites, Arg155-Ser[156] and Arg284-Thr[285], generating prethrombin 1 (Pre 1) and fragment 1 and generating Pre 2Ј and fragment 2, respectively
Summary
Protein Purification and Characterization—ProT was purified from human plasma (30). ␣-Thrombin was active site-titrated by previously described methods (30). Met-SC-(1–325)-labeled ProT complex was concentrated and dissociated by dialysis against Buffer A with 3 M NaSCN but without polyethylene glycol 8000. Trace amounts of Met-SC-(1–325)-His6-labeled ProT complex were removed by fast protein liquid chromatography gel filtration on Superdex 200 HR 10/30 in Buffer B. The concentration of residual active ProT was determined by chromogenic substrate assay of 25 nM labeled ProT after incubation with a 5-fold excess of SC-(1–325) for 20 min at 25 °C, relative to a known concentration of the native SC-(1–325)1⁄7ProT complex. Together with the direct titration data of [OG]FPR-ProT with SC-(1–325), the data were fit simultaneously by the cubic equation for tight competitive binding of SC-(1–325) to labeled and native ProT to determine the dissociation constant and stoichiometry for the native SC-(1–325)1⁄7ProT interaction (38, 39). Because the complex-dependent proteolysis described below is minimized at the concentrations used in the kinetic studies, there was no variation of the initial rates beyond the 20-min preincubation time
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