title: Protein engineering of the tissue inhibitor of metalloproteinase 1 (TIMP-1) inhibitory domain. In search of selective matrix metalloproteinase inhibitors.
abstract: Studies of the structural basis of the interactions of tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) may provide clues for designing MMP-specific inhibitors. In this paper we report combinations of mutations in the major MMP-binding region that enhance the specificity of N-TIMP-1. Mutants with substitutions for residues 4 and 68 were characterized and combined with previously studied Thr(2) mutations to generate mutants with improved selectivity or binding affinity to specific MMPs. Some combinations of mutations had non-additive effects on DeltaG of binding to MMPs, suggesting interactions between subsites in the reactive site. The T2L/V4S mutation generates an inhibitor that binds to MMP-2 20-fold more tightly than to MMP-3(DeltaC) and over 400-fold more tightly than to MMP-1. The T2S/V4A/S68Y mutant is the strongest inhibitor for stromelysin-1 among all mutants characterized to date, with an apparent K(i) for MMP-3(DeltaC) in the picomolar range. A third mutant, T2R/V4I, has no detectable inhibitory activity for MMP-1 but is an effective inhibitor of MMP-2 and -3. These selective TIMP variants may provide useful tools for investigation of biological roles of specific MMPs and for possible therapy of MMP-related diseases.
authors.affiliation: Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, Florida 33431, USA.
authors.forename: Shuo
authors.initials: S
authors.lastname: Wei
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: Linda
authors.initials: L
authors.lastname: Chung
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagase
authors.forename: Keith
authors.initials: K
authors.lastname: Brew
mesh_terms: D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000737:chemistry
chemical_list: D019715:Tissue Inhibitor of Metalloproteinase-1; D013912:Threonine; D012694:Serine; D020782:Matrix Metalloproteinases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D014633:Valine