Unveiling the surface epitopes that render tissue inhibitor of metalloproteinase-1 inactive against membrane type 1-matrix metalloproteinase.
Membrane type 1-matrix metalloproteinase (MT1-MMP) is a zinc-dependent, membrane-associated endoproteinase of the metzincin family. The enzyme regulates extracellular matrix remodeling and is capable of cleaving a wide variety of transmembrane proteins. The enzymatic activity of MT1-MMP is regulated by endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMP). To date, four variants of mammalian TIMP have been identified. Whereas TIMP-2-4 are potent inhibitors against MT1-MMP, TIMP-1 displays negligible inhibitory activity against the enzyme. The rationale for such selectivity is hitherto unknown. Here we identify the surface epitopes that render TIMP-1 inactive against MT1-MMP. We show that TIMP-1 can be transformed into an active inhibitor against MT1-MMP by the mutation of a single residue, namely threonine 98 to leucine (T98L). The resultant mutant displayed inhibitory characteristics of a typical slow, tight binding inhibitor. The potency of the mutant could be further enhanced by the introduction of valine 4 to alanine (V4A) and proline 6 to valine (P6V) mutations. Indeed, the inhibitory profile of the triple mutant (V4A/P6V/T98L) is indistinguishable from those of other TIMPs. Our findings suggest that threonine 98 is critical in initiating MMP binding and complex stabilization. Our findings also provide a potential mechanistic explanation for MMP-TIMP selectivity.
Lee
Meng-Huee
MH
Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Cambridge University, Cambridge CB2 2XY, United Kingdom.
Rapti
Magdalini
M
Murphy
Gillian
G
D000595:Amino Acid Sequence; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000737:chemistry / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000737:chemistry / Q000235:genetics; D019714:Tissue Inhibitor of Metalloproteinases / Q000737:chemistry / Q000235:genetics; D000097792:Tissue Inhibitor of Metalloproteinase-4
D000939:Epitopes; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D011480:Protease Inhibitors; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D019717:Tissue Inhibitor of Metalloproteinase-3; D019714:Tissue Inhibitor of Metalloproteinases; D000097792:Tissue Inhibitor of Metalloproteinase-4