Conformational Changes in Thrombin When Complexed by Serpins
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abstract
Thrombin possesses two positively charged surface domains, termed exosites, that orient substrates and inhibitors for reaction with the enzyme. Because the exosites also allosterically modulate thrombin's activity, we set out to determine whether the structure or function of the exosites changes when 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 integrity of exosite 1, no binding was detected when thrombin was complexed with heparin cofactor II or alpha(1)-antitrypsin (M358R), and the peptide exhibited a 55-fold lower affinity for the thrombin-antithrombin complex than for thrombin. Bound peptide or HD-1, an exosite 1-binding DNA aptamer, was displaced from thrombin by each of the three serpins. Thrombin binding to fibrin also was abrogated when the enzyme was complexed with serpins. These data reveal that, regardless of the initial mode of interaction, the function 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 by serpins, because interaction with heparin or an exosite 2-directed DNA aptamer was only modestly altered. The disorganization of exosite 1 that occurs when thrombin is complexed by serpins is consistent with results of protease sensitivity studies and crystallographic analysis of a homologous enzyme-serpin complex.
