Crystal structure of thrombin-ecotin reveals conformational changes and extended interactions.

The protease inhibitor ecotin fails to inhibit thrombin despite its broad specificity against serine proteases. A point mutation (M84R) in ecotin results in a 1.5 nM affinity for thrombin, 10(4) times stronger than that of wild-type ecotin. The crystal structure of bovine thrombin is determined in complex with ecotin M84R mutant at 2.5 A resolution. Surface loops surrounding the active site cleft of thrombin have undergone significant structural changes to permit inhibitor binding. Particularly, the insertion loops at residues 60 and 148 in thrombin, which likely mediate the interactions with macromolecules, are displaced when the complex forms. Thrombin and ecotin M84R interact in two distinct surfaces. The loop at residue 99 and the C-terminus of thrombin contact ecotin through mixed polar and nonpolar interactions. The active site of thrombin is filled with eight consecutive amino acids of ecotin and demonstrates thrombin's preference for specific features that are compatible with the thrombin cleavage site: negatively charged-Pro-Val-X-Pro-Arg-hydrophobic-positively charged (P1 Arg is in bold letters). The preference for a Val at P4 is clearly defined. The insertion at residue 60 may further affect substrate binding by moving its adjacent loops that are part of the substrate recognition sites.