Canonical inhibitor-like interactions explain reactivity of α1-proteinase inhibitor Pittsburgh and antithrombin with proteinases

The serpin antithrombin is a slow thrombin inhibitor that requires heparin to enhance its reaction rate. In contrast, alpha(1)-proteinase inhibitor (alpha(1)PI) Pittsburgh (P1 Met --> Arg natural variant) inhibits thrombin 17 times faster than pentasaccharide heparin-activated antithrombin. We present here x-ray structures of free and S195A trypsin-bound alpha(1)PI Pittsburgh, which show that the reactive center loop (RCL) possesses a canonical conformation in the free serpin that does not change upon binding to S195A trypsin and that contacts the proteinase only between P2 and P2'. By inference from the structure of heparin cofactor II bound to S195A thrombin, this RCL conformation is also appropriate for binding to thrombin. Reaction rates of trypsin and thrombin with alpha(1)PI Pittsburgh and antithrombin and their P2 variants show that the low antithrombin-thrombin reaction rate results from the antithrombin RCL sequence at P2 and implies that, in solution, the antithrombin RCL must be in a similar canonical conformation to that found here for alpha(1)PI Pittsburgh, even in the nonheparin-activated state. This suggests a general, limited, canonical-like interaction between serpins and proteinases in their Michaelis complexes.