Structure of the complex of trypsin with a highly potent synthetic inhibitor at 0.97 Å resolution

The structure of the complex formed between bovine β-trypsin and the highly potent synthetic inhibitor 2-{3′-[5′-methoxy-2′-(N-p-diaminomethylphenyl)amido]-1′-pyrido}-5-(N-2″-t-butylethanol)amidobenzoic acid (C₂₈H₃₂N₅O₆) has been determined at 0.97 Å resolution. X-ray intensity data were collected to 0.97 Å under cryocooled conditions. The structure was refined anisotropically using REFMAC5 and SHELX-97 to R_cryst factors of 13.4 and 12.6% and R_free factors of 15.7 and 16.3%, respectively. Several regions of the main chain and side chains that have not been previously observed were clearly defined in the present structure. H atoms are indicated as significant peaks in an |F_o − F_c| difference map, which accounts for an estimated 35% of all H atoms at the 2.5σ level. The C, N and O atoms are definitively differentiated in the electron-density maps. The amido part of the inhibitor occupies the specificity pocket and the remainder fills the remaining part of the ligand-binding cleft and interacts with the enzyme through an extensive network of hydrogen bonds. The inhibitor distorts the stereochemistry of the catalytic triad, Ser195–His57–Asp102, thereby blocking the proton-relay process of the active site by preventing the formation of the crucial hydrogen bond between His57 N^δ1 and Asp102 O^δ1.