RESUMO
BACKGROUND: For many enzymes, substrate specificity is directed by secondary binding sites (exosites) that are remote from the active site. Peptide inhibition studies of protein-protein interactions are useful to identify exosite functions. OBJECTIVE: To develop an approach to manipulate these exosites using ligand-directed covalent modification of the enzyme. METHOD: To demonstrate this strategy, we have engineered an exosite-deficient variant of human plasma-derived thrombin (FIIa) . Desulfato-hirugen (Hir(55-65)) analogs were synthesized with a fluorescent label, photocrosslinker, and an optional cleavable linker conjugated to the N-terminus of the peptide, specifically fluorescein-benzoyl-phenylalanyl-(Fl-bF-)glycyl-Hir(55-65), Fl-bF-mercaptopropionyl-Hir(55-65) and Fl-bF-lactyl-Hir(55-65) were synthesized. Each analog was bound and photocrosslinked to FIIa, and the resulting covalent complex was purified. RESULTS: This modified enzyme, FIIa-Hir(55-65), hydrolyzed small substrates as efficiently as native FIIa, but was significantly inhibited in fibrinogen clotting and in thrombomodulin-mediated PC activation, implying that the active site was unaffected by labeling but exosite I was blocked. In addition, this approach was used to transfer a fluorescein label from the exosite I binding peptide Hir(55-65) to a site proximal to but not obstructing exosite I. The activity of this fluorescently labeled FIIa (Fl-FIIa) could be inhibited by unlabeled Hir(55-65), suggesting that exosite I is unmodified. Importantly, this interaction could be followed spectroscopically by fluorescence, demonstrating that the exosite I proximal probe can be used to monitor specific ligand binding interactions. CONCLUSION: Our results show that exosites of clotting factors (e.g. thrombin) can be specifically inhibited and labeled with fluorescent reporters. This novel technology may have broad applicability for studies of protein-protein interactions that regulate coagulation.
