title: Mapping and characterization of the functional epitopes of tissue inhibitor of metalloproteinases (TIMP)-3 using TIMP-1 as the scaffold: a new frontier in TIMP engineering.
abstract: Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) is responsible for the release of TNF-alpha, a potent proinflammatory cytokine associated with many chronic debilitating diseases such as rheumatoid arthritis. Among the four variants of mammalian tissue inhibitor of metalloproteinases (TIMP-1 to -4), TACE is specifically inhibited by TIMP-3. We set out to delineate the basis for this specificity by examining the solvent accessibility of every epitope on the surface of a model of the truncated N-terminal domain form of TIMP-3 (N-TIMP-3) in a hypothetical complex with the crystal structure of TACE. The epitopes suspected of interacting with TACE were systematically transplanted onto N-TIMP-1. We succeeded in transforming N-TIMP-1 into an active inhibitor for TACE (K(i)(app) 15 nM) with the incorporation of Ser4, Leu67, Arg84, and the TIMP-3 AB-loop. The combined effects of these epitopes are additive. Unexpectedly, introduction of "super-N-TIMP-3" epitopes, defined in our previous work, only impaired the affinity of N-TIMP-1 for TACE. Our mutagenesis results indicate that TIMP-3-TACE interaction is a delicate process that requires highly refined surface topography and flexibility from both parties. Most importantly, our findings confirm that the individual characteristics of TIMP could be transplanted from one variant to another.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
authors.forename: Meng-Huee
authors.initials: MH
authors.lastname: Lee
authors.forename: Klaus
authors.initials: K
authors.lastname: Maskos
authors.forename: Vera
authors.initials: V
authors.lastname: Knäuper
authors.forename: Philippa
authors.initials: P
authors.lastname: Dodds
authors.forename: Gillian
authors.initials: G
authors.lastname: Murphy
mesh_terms: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D008666:Metalloendopeptidases / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D016415:Sequence Alignment; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000276:immunology*; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000276:immunology*
chemical_list: D000939:Epitopes; D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human