Thermolysin in the absence of substrate has an open conformation

The bacterial neutral proteases have been proposed to undergo hinge-bending during their catalytic cycle. However, in thermolysin, the prototypical member of the family, no significant conformational change has been observed. The structure of thermolysin has now been determined in a new crystal form that for the first time shows the enzyme in the absence of a ligand bound in the active site. This is shown to be an `open' form of the enzyme. The relative orientation of the two domains that define the active-site cleft differ by a 5° rotation relative to their positions in the previously studied ligand-bound `closed' form. Based on structural comparisons, kinetic studies on mutants and molecular-dynamics simulations, Gly78 and Gly135-Gly136 have previously been suggested as two possible hinge regions. Comparison of the `open' and `closed' structures suggests that neither of the proposed hinge regions completely accounts for the observed displacement. The concerted movement of a group of side chains suggested to be associated with the hinge-bending motion is, however, confirmed.