Conformational changes in thrombin when complexed by serpins

Thrombin possesses two positively charged surface domains, termed exosites, that orient substrates and inhibitors for reaction with the enzyme. Becaus e the exosites also allosterically modulate thrombin's activity, we set out to determine whether the structure or function of the exosites changes whe n thrombin forms complexes with antithrombin, heparin cofactor II, or alpha (1)-antitrypsin (M358R), serpins that utilize both, one, or neither of the exosites, respectively. Using a hirudin-derived peptide to probe the integ rity of exosite 1, no binding was detected when thrombin was complexed with heparin cofactor II or alpha (1)-antitrypsin (M358R), and the peptide exhi bited a 55-fold lower affinity for the thrombin-antithrombin complex than f or thrombin. Bound peptide or HD-1, an exosite 1-binding DNA aptamer, was d isplaced from thrombin by each of the three serpins. Thrombin binding to fi brin also was abrogated when the enzyme was complexed with serpins. These d ata reveal that, regardless of the initial mode of interaction, the functio n of exosite 1 is lost when thrombin is complexed by serpins. In contrast, the integrity of exosite 2 is largely retained when thrombin is complexed b y serpins, because interaction with heparin or an exosite 2-directed DNA ap tamer was only modestly altered. The disorganization of exosite 1 that occu rs when thrombin is complexed by serpins is consistent with results of prot ease sensitivity studies and crystallographic analysis of a homologous enzy me-serpin complex.