Crystal structure of cancer chemopreventive Bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 angstrom resolution. Structural basisof Janus-faced serine protease inhibitor specificity

Understanding molecular recognition on a structural basis is an objective w ith broad academic and applied significance. In the complexes of serine pro teases and their proteinaceous inhibitors, recognition is governed mainly b y residue P1 in accord with primary serine protease specificity. The bifunc tional soybean Bowman-Birk inhibitor (sBBI) should, therefore, interact at LysI16 (subdomain 1) with trypsin and at LeuI43 (subdomain 2) with chymotry psin. In contrast with this prediction, a 2:1 assembly with trypsin was obs erved in solution and in the crystal structure of sBBI in complex with tryp sin, determined at 2.3 Angstrom resolution by molecular replacement. Striki ngly, P1LeuI43 of sBBI was fully embedded into the S-1 pocket of trypsin in contrast to primary specificity. The triple-stranded beta-hairpin unique t o the BBI-family and the surface loops surrounding the active site of the e nzyme formed a protein-protein-interface far extended beyond the primary co ntact region. Polar residues, hydrophilic bridges and weak hydrophobic cont acts were predominant in subdomain 1, interacting specifically with trypsin . However, close hydrophobic contacts across the interface were characteris tic of subdomain 2 reacting with both trypsin and chymotrypsin. A Met27Ile replacement shifted the ratio with trypsin to the predicted 1:1 ratio. Thus , the buried salt-bridge responsible for trypsin specificity was stabilised in a polar, and destabilised in a hydrophobic, environment. This may be us ed for adjusting the specificity of protease inhibitors for applications su ch as insecticides and cancer chemopreventive agents. (C) 2000 Academic Pre ss.