title: Tailoring tissue inhibitor of metalloproteinases-3 to overcome the weakening effects of the cysteine-rich domains of tumour necrosis factor-alpha converting enzyme.
abstract: Tumour necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) is a membrane-anchored, multiple-domain zinc metalloproteinase responsible for the release of the potent pro-inflammatory cytokine, TNF-alpha. The extracellular part of the active enzyme is composed of a catalytic domain and several cysteine-rich domains. Previously, we reported that these cysteine-rich domains significantly weakened the inhibitory potency of the N-terminal-domain form of tissue inhibitor of metalloproteinases-3 (N-TIMP-3). In the present paper, we describe a novel strategy developed to overcome this weakening effect. We have engineered a new generation of N-TIMP-3 mutants that are capable of withstanding the repulsion of the cysteine-rich domains by the formation of electrostatic bonds with the catalytic domain of the enzyme. These N-TIMP-3 mutants displayed markedly improved binding affinity with the soluble extracellular domain form of recombinant TACE. With K (i) (app) values of <0.1 nM, these mutants were dramatically better than the wild-type N-TIMP-3 [K (i) (app) 1.7 nM]. We accounted for this by proposing that Glu(31), an acidic residue situated at the base of the AB-loop of N-TIMP-3, is drawn into contact with Lys(315), a prominent basic residue adjacent to the TACE catalytic site. The mutagenesis strategy involved reorientation of the edge of N-TIMP-3; in particular, the beta-strand A where Glu(31) was located. Further expression of one of the mutants, Lys(26/27/30/76)-->Glu, in a mammalian expression system confirmed that TIMP-3 associates with the extracellular matrix via its C-terminal domain.
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: Philippa
authors.initials: P
authors.lastname: Dodds
authors.forename: Vandana
authors.initials: V
authors.lastname: Verma
authors.forename: Klaus
authors.initials: K
authors.lastname: Maskos
authors.forename: Vera
authors.initials: V
authors.lastname: Knäuper
authors.forename: Gillian
authors.initials: G
authors.lastname: Murphy
mesh_terms: D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003545:Cysteine; D007700:Kinetics; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D009101:Multiple Myeloma; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D017384:Sequence Deletion; D055672:Static Electricity; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000737:chemistry* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
chemical_list: D011994:Recombinant Proteins; D019717:Tissue Inhibitor of Metalloproteinase-3; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606463:Adam17 protein, mouse; D003545:Cysteine