Protein engineering of the tissue inhibitor of metalloproteinase 1 (TIMP-1) inhibitory domain. In search of selective matrix metalloproteinase inhibitors.
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.
Wei
Shuo
S
Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, Florida 33431, USA.
Chen
Ying
Y
Chung
Linda
L
Nagase
Hideaki
H
Brew
Keith
K
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
D019715:Tissue Inhibitor of Metalloproteinase-1; D013912:Threonine; D012694:Serine; D020782:Matrix Metalloproteinases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D014633:Valine