Manipulation of thrombin exosite I, by ligand-directed covalent modification

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.

To develop an approach to manipulate these exosites using ligand-directed covalent modification of the enzyme.

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.

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.

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.