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.
