The thrombin E192Q-BPTI complex reveals gross structural rearrangements: implications for the interaction with antithrombin and thrombomodulin

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Abstract
Previous crystal structures of thrombin indicate that the 60‐insertion loop is a rigid moiety that partially occludes the active site, suggesting that this structural feature plays a decisive role in restricting thrombin‘s specificity. This restricted specificity is typified by the experimental observation that thrombin is not inhibited by micromolar concentrations of basic pancreatic trypsin inhibitor (BPTI). Surprisingly, a single atom mutation in thrombin (E192Q) results in a 10−8 M affinity for BPTI. The crystal structure of human thrombin mutant E192Q has been solved in complex with BPTI at 2.3 Å resolution. Binding of the Kunitz inhibitor is accompanied by gross structural rearrangements in thrombin. In particular, thrombin's 60‐loop is found in a significantly different conformation. Concomitant reorganization of other surface loops that surround the active site, i.e. the 37‐loop, the 148‐loop and the 99‐loop, is observed. Thrombin can therefore undergo major structural reorganization upon strong ligand binding. Implications for the interaction of thrombin with antithrombin and thrombomodulin are discussed.