Structural analysis of the primary specificity of thrombin.

Abstract

Background: Thrombin has dual trypsin-like and chymotrypsin-like specificity: it prefers substrates carrying Arg at the site of cleavage (P1) in the activation peptide because of the presence of D189 in the primary specificity (S1) site but can also cleave substrates carrying Phe at P1.

Objectives: Explore the structural basis of the P1-S1 interaction with mutants of D189 and substrates carrying different residues at P1.

Methods: X-ray crystallography is used to solve the structures of thrombin wild-type and mutants D189A, D189F, and D189K bound to the irreversible inhibitors H-D-Phe-Pro-Arg-CH₂Cl (FPRck), H-D-Phe-Pro-Phe-CH₂Cl (FPFck), and H-D-Phe-Pro-Gln-CH₂Cl (FPQck).

Results: X-ray structures of thrombin wild-type and mutants D189A, D189F, and D189K bound to FPRck, FPFck, and FPQck are solved at high resolutions, from 1.2 Å to 2.5 Å. Mutations of D189 do not affect the stability of free thrombin but shift the substrate preference from Arg to Phe at P1 and the stability of the inhibited complex from FPRck to FPFck. The structures reveal the flexibility of the S1 site in accommodating side chains of different chemical properties to optimize the P1-S1 interaction. In the D189K-FPRck complex, the inhibitor is trapped in an intermediate state, covalently bound only to the catalytic H57 and with the Arg side chain positioned outside of the active site.

Conclusions: The S1 site of thrombin features significant structural flexibility and can support the replacement of D189 with Ala, Phe, or even Lys. The results set the stage for future studies aimed at re-engineering primary specificity in thrombin and other trypsin-like proteases.