STRUCTURE OF THE HIRULOG-3 THROMBIN COMPLEX AND NATURE OF THE S' SUBSITES OF SUBSTRATES AND INHIBITORS

The X-ray crystallographic structure of the human alpha-thrombin complex with hirulog 3 (a potent, noncleavable hirudin-based peptide of the "hirulog" class containing a beta-homoarginine at the scissile bond), which is isomorphous with that of the hirugen-thrombin crystal structure, was solved at 2.3-angstrom resolution by starting with a model for thrombin derived from the hirugen-thrombin complex and was refined by restrained least squares methods (R = 0.132). Residues of hirulog 3 were well-defined in the electron density, which included most of the pentaglycine linker and the C-terminal helical turn that was disordered in a related structure of thrombin with hirulog 1. The interactions of D-Phe1'-Pro2'-beta-homoArg3' with the active site of thrombin were essentially identical to those of related structures of PPACK- (D-Phe-Pro-Arg chloromethyl ketone) and hirulog 1-thrombin, with the guanidinium function of the arginyl P1 residue forming a hydrogen-bonding ion pair with Asp189 of the S1 site. A noticeable shift in the CA atom of beta-homoArg3' due to the methylene insertion displaces the scissile bond from attack by Ser195, thus imparting proteolytic stability to the beta-homoArg hirulog derivative. Resolution of the pentaglycine spacer, linking N- and C-terminal functional domains into a single oligopeptide bivalent inhibitor, permitted delineation of corresponding S' subsites of thrombin. The position of Gly4' (P1') is stabilized by three hydrogen bonds with His57, Lys60F, and Ser195, while the conformational angles are maintained in a strained, nonallowed configuration for non-glycyl amino acids. The S1' site inferred from the positioning of the CA atom of Gly4' is a small cavity lined by amino acids of the back side of the apolar S2 pocket (His57,Tyr60A, Trp60D) and the side chain of Lys60F. This putative S1' subsite can accommodate mall polar amino acid side chains as observed from P1' amino acids in a number of natural thrombin substrates. A larger surface cavity appears to define the S2'-S3' sites, lined by Lys60F, Leu41, Phe60H, and Glu39, as determined by the positioning of Gly5'-Gly6' of hirulog 3. Not withstanding the absence of tyrosine sulfation in hirulog 3, Pro60'-Leu64' interacting with the anion-binding exo site of fibrinogen recognition are ordered in a 3(10) helical turn. As with hirugen-and hirudin-thrombin complexes, this binding interaction is predominantly hydrophobic. In summary, the structural determination and analysis of the thrombin complex with hirulog 3 provide complete molecular detail for hirulog-thrombin interactions. By analogy with thrombin-substrate interactions, the hirulog structure has defined S' subsites that appear to govern further specificity in thrombin physiologic actions. The fact that Asn53'-Gly54' are disordered in all the structures of thrombin complexed with hirudin C-terminal peptides is most likely the result of nonenzymatic deamidation of the asparagine residue.