We have previously reported the crystal structure of truncated human collagenase (domain II) complexed with a low molecular weight inhibitor. Attempts to crystallize full-length active collagenase (i.e. domain II + III) have been hindered by autoproteolysis at the domain II/III junction at high protein concentrations. To overcome this problem, we have generated an inactive enzyme via a H149-->L,D151-->N double substitution which displaces the non-catalytic zinc atom, and shown that the altered collagenase is unable to cleave a synthetic substrate. We have also generated an 1251-->S substitution at the domain II/III junction and demonstrate an increased resistance to proteolysis compared to wild-type collagenase.