The Methyl Group of Nα(Me)Arg-containing Peptides Disturbs the Active-site Geometry of Thrombin, Impairing Efficient Cleavage

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-Nα(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with Ki values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (Ki=37pM), containing an Nα(Me)Arg-Thr bond, was crystallized in complex with human α-thrombin. X-ray data were collected to 1.8 Å resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The Nα(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 Oγ atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 Oγ are separated from each other, impairing the nucleophilic attack of the Ser195 Oγ toward the Nα(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.