The Conformation of Thermolysin

Abstract An improved electron density map has been obtained for the thermostable protease thermolysin. The map, calculated at a nominal resolution of 2.3 A, was based on the three heavy atom isomorphs described previously and on data for the zinc-free and europium-substituted enzyme. The zinc-free and native enzymes are closely isomorphous, and the difference in electron density between them shows that the only structural changes which occur on the removal of the zinc ion are slight adjustments in residues in the immediate vicinity of the metal ion. The approximate conformation of most of the thermolysin molecule is revealed without ambiguity, and preliminary atomic coordinates are presented. Some aspects of the molecular stereochemistry are discussed, including tertiary folding, helices, turns, and calcium binding sites. The peptide dihedral angles for most of the residues lie within or near the allowed regions for an empirical hard sphere model, but the conformations of a few residues lie in disallowed regions. There are several indications that the angle τ at the α carbon atom must be significantly greater than the tetrahedral for a number of residues. With the possible exception of the four calcium binding sites, there is nothing particularly unusual in the thermolysin conformation to which one might attribute the thermostability of the molecule. It is suggested that the enhanced stability of thermostable proteins relative to thermolabile ones cannot be attributed to a common determinant such as metal ion or hydrophobic stabilization, but in a given instance may be due to rather subtle differences in hydrophobic character, metal binding, hydrogen bonding, ionic interactions, or a combination of all of these.