title: The catalytic domain of activated collagenase I (MMP-1) is absolutely required for interaction with its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1).
abstract: Here, we describe the production of recombinant human tissue inhibitor of metalloproteinases-1 (rTIMP-1) and wild-type and mutant human collagenase type I (rMMP-1) proteins in SF9 cells by the baculovirus expression 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 enzymatic activity upon cleavage of the prodomain by treatment with trypsin or 4-aminophenylmercuric acetate. Enzyme activity of both proteins can 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 stable 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 rTIMP to determine the structural requirements of MMP-1 to form complexes 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 to form complexes with TIMP. Neither mutation of His199, nor deletion mutants des-(161-228)-MMP-1 and des-(161-228/248-450)-MMP-1, interact with TIMP. This demonstrates that the C-terminal hemopexin domain of MMP-1, in contrast to the corresponding regions of gelatinase A and gelatinase B, does not interact with TIMP-1. In summary, we have shown that the integrity of the catalytic domain of MMP-1 and its ability to bind Zn2+ is absolutely required for complex formation with TIMP-1, which further underlines the importance of this region for proper regulation of enzymatic activity of MMP-1.
authors.affiliation: Deutsches Krebsforschungszentrum Heidelberg, Abteilung Signaltransduktion und Wachstumskontrolle, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Vallon
authors.forename: R
authors.initials: R
authors.lastname: Müller
authors.forename: D
authors.initials: D
authors.lastname: Moosmayer
authors.forename: E
authors.initials: E
authors.lastname: Gerlach
authors.forename: P
authors.initials: P
authors.lastname: Angel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002478:Cells, Cultured; D017364:Collagenases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020781:Matrix Metalloproteinase 1; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics; D019714:Tissue Inhibitor of Metalloproteinases
chemical_list: D006023:Glycoproteins; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D017364:Collagenases; D020781:Matrix Metalloproteinase 1