THE CATALYTIC DOMAIN OF ACTIVATED COLLAGENASE-I (MMP-1) IS ABSOLUTELYREQUIRED FOR INTERACTION WITH ITS SPECIFIC INHIBITOR, TISSUE INHIBITOR OF METALLOPROTEINASES-1 (TIMP-1)

Here, we describe the production of recombinant human tissue inhibitor of metalloproteinases-l (rTIMP-1) and wild-type and mutant human coll agenase type I (rMMP-1) proteins in SF9 cells by the baculovirus expre ssion system. Wild-type MMP-1, as well as the MMP-1 mutant lacking the C-terminal hemopexin-like domain [des-(248-450)-MMP-1], exhibit enzym atic activity upon cleavage of the pro-domain by treatment with trypsi n or 4-aminophenylmercuric acetate. Enzyme activity of both proteins c an be inhibited by addition of rTIMP. Deletion of the complete active- site [des-(161-228)-MMP-1] within the catalytic domain, or mutation of a single His residue of the Zn2+ binding domain (His199), generates s table forms of MMP-1 proteins which are unable to digest collagen type I or beta-casein. In addition to co-immunoprecipitation analysis, we have established a rapid and sensitive ELISA assay using immobilized r TIMP to determine the structural requirements of MMP-1 to form complex es with its inhibitor. Only the activated and not the latent forms of wild-type and C-terminal mutant des-(248-450)MMP-1 proteins are able t o form complexes with TIMP. Neither mutation of His199, nor deletion m utants des-(161-228)-MMP-1 and des-(161-228/248-450)-MMP-1, interact w ith TIMP. This demonstrates that the C-terminal hemopexin domain of MM P-1, in contrast to the corresponding regions of gelatinase A and gela tinase B, does not interact with TIMP-1. In summary, we have shown tha t the integrity of the catalytic domain of MMP-1 and its ability to bi nd Zn2+ is absolutely required for complex formation with TIMP-1, whic h further underlines the importance of this region for proper regulati on of enzymatic activity of MMP-1.