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

The structure of the complex formed between bovine beta-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 angstrom resolution. X-ray intensity data were collected to 0.97 angstrom 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 vertical bar F-o - F-c vertical bar difference map, which accounts for an estimated 35% of all H atoms at the 2.5 sigma 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-delta 1 and Asp102 O-delta 1.