The X-ray crystal structure of the human α-thrombin-hirunorm
IV complex has been determined at 2.5 Å resolution,
and refined to an R-factor of 0.173. The structure
reveals an inhibitor binding mode distinctive of a true
hirudin mimetic, which justifies the high inhibitory potency
and the selectivity of hirunorm IV. This novel inhibitor,
composed of 26 amino acids, interacts through the N-terminal
end with the α-thrombin active site in a nonsubstrate
mode, and binds specifically to the fibrinogen recognition
exosite through the C-terminal end. The backbone of the
N-terminal tripeptide Chg1"-Arg2"-2Nal3"
(Chg, cyclohexyl-glycine; 2Nal, β-(2-naphthyl)-alanine)
forms a parallel β-strand to the thrombin main-chain
segment Ser214–Gly216. The Chg1" side chain
occupies the S2 site, Arg2" penetrates into the S1
specificity site, while the 2Nal3" side chain occupies
the aryl binding site. The Arg2" side chain enters
the S1 specificity pocket from a position quite apart from
the canonical P1 site. This notwithstanding, the Arg2"
side chain establishes the typical ion pair with the carboxylate
group of Asp189.