In:
Biochemical Journal, Portland Press Ltd., Vol. 371, No. 2 ( 2003-04-15), p. 369-376
Abstract:
Tumour necrosis factor-α (TNF-α) converting enzyme (TACE) is a membrane-anchored, multiple-domain zinc metalloproteinase responsible for the release of the potent pro-inflammatory cytokine, TNF-α. 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 Ki (app) values of & lt;0.1nM, these mutants were dramatically better than the wild-type N-TIMP-3 [Ki (app) 1.7nM]. We accounted for this by proposing that Glu31, an acidic residue situated at the base of the AB-loop of N-TIMP-3, is drawn into contact with Lys315, 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 β-strand A where Glu31 was located. Further expression of one of the mutants, Lys26/27/30/76→Glu, in a mammalian expression system confirmed that TIMP-3 associates with the extracellular matrix via its C-terminal domain.
Type of Medium:
Online Resource
ISSN:
0264-6021
,
1470-8728
Language:
English
Publisher:
Portland Press Ltd.
Publication Date:
2003
detail.hit.zdb_id:
1473095-9
SSG:
12
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