Tailoring tissue inhibitor of metalloproteinases-3 to overcome the weakening effects of the cysteine-rich domains of tumour necrosis factor-alpha converting enzyme.
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.[1]References
- Tailoring tissue inhibitor of metalloproteinases-3 to overcome the weakening effects of the cysteine-rich domains of tumour necrosis factor-alpha converting enzyme. Lee, M.H., Dodds, P., Verma, V., Maskos, K., Knäuper, V., Murphy, G. Biochem. J. (2003) [Pubmed]
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