The 0.78 Å Structure of a Serine Protease:  Bacillus lentus Subtilisin^,

Abstract

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc 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 A 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.

Hydrogen atoms in proteins: Positions and dynamics

“…[4][5][6]. Even protonation states of amino acid side chains can be determined with adequate highquality x-ray data sets (7).…”

The Crystal Structure of a Novel, Inactive, Lysine 49 PLA2 from Agkistrodon acutus Venom

“…For example, in the acutohaemolysin case, each atom can be described by nine parameters, including three coordinates and six thermal factors, that express the anisotropic nature of the thermal motion, and this permits the solvent molecules to be divided into two hydration shells. In addition, atomic resolution difference electron density maps allow the positions of a large number of hydrogen atoms, including those involved in interactions among active-site residues, to be located (15). Moreover, alternative or multiple conformations of some disordered residues can be resolved and modeled.…”

A branched N-linked glycan at atomic resolution in the 1.12 Å structure of rhamnogalacturonan acetylesterase

“…In the otherwise structurally unrelated serine protease subtilisin (Kuhn et al, 1998), the nucleophile is located on a type I -turn as in the SGNH-hydrolases. Subtilisin does not have an Asp in the same position as the SGNH hydrolases, but a similar hydrogen bond is formed between the Nu À 1 backbone carboxyl group and the Nu + 1 amide group.…”

“…not possible to perform completely unrestrained re®nement despite a relatively high ratio of observations to parameters (5.6:1) (Sandalova et al, 1999). In the 0.78 A Ê structure of Bacillus lentus subtilisin, an unrestrained re®nement (10:1 ratio of observations to parameters) was carried out without problems however (Kuhn et al, 1998). The target values for the geometrical restraints are usually taken from an analysis of small-molecule X-ray structures from the Cambridge Structural Database performed by Engh & Huber (1991).…”