The 0.78 Å structure of a serine protease:: Bacillus lentus subtilisin

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 Angstrom. The refined model coordinates have a rms deviation of 0.22 Angstrom relative to the same structure determined at room temperature and 2.0 Angstrom resolution. Several regions of main-chain and side-chain disorder have been identified for 21 our of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the F-o - F-c difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 Angstrom and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.