title: Mutational study of the amino-terminal domain of human tissue inhibitor of metalloproteinases 1 (TIMP-1) locates an inhibitory region for matrix metalloproteinases.
abstract: A bacterial expression system for the inhibitory N-terminal domain of human tissue inhibitor of metalloproteinases 1 (N-TIMP-1) (Huang, W., Suzuki, K., Nagase, H., Arumugam, S., Van Doren, S. R., and Brew, K. (1996) FEBS Lett. 384, 155-161) has been used to produce 20 single- and double-site mutants that probe the roles of different residues in its inhibitory action on metalloproteinases. Mutations that produce the largest increases in the Ki for a C-terminally truncated form of stromelysin 1, MMP-3(DeltaC), but do not disturb the conformation involve substitutions of residues that are located in a ridge that is centered around the disulfide bond between Cys1 and Cys70. Specific residues that have a large influence on activity include Cys1, Thr2, Met66, Val69, and Cys70. Of the mutations introduced, the greatest functional disturbances, reflected in Ki increases of 2-4 orders of magnitude, are generated by changes that disrupt the Cys1-Cys70 disulfide bond and by substitution of Ala for Thr2. Most mutations that perturb the interaction with MMP-3 have parallel effects on the affinity of N-TIMP-1 for MMP-1 (interstitial collagenase) and MMP-2 (gelatinase A). However, the Thr2 to Ala mutation produces an inhibitor that is 17-fold more effective against MMP-3 than MMP-1, suggesting that it is feasible to engineer TIMP-1 variants that are more specifically targeted to selected matrix metalloproteinases. The reactive site identified by these studies is a structurally constrained but elongated region of TIMP that can fit the matrix metalloproteinase substrate-binding site.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
authors.forename: W
authors.initials: W
authors.lastname: Huang
authors.forename: Q
authors.initials: Q
authors.lastname: Meng
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
authors.forename: H
authors.initials: H
authors.lastname: Nagase
authors.forename: K
authors.initials: K
authors.lastname: Brew
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D017364:Collagenases / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism; D005818:Genetic Engineering; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D020781:Matrix Metalloproteinase 1; D020778:Matrix Metalloproteinase 2; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D019714:Tissue Inhibitor of Metalloproteinases
chemical_list: D006023:Glycoproteins; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1