Tailoring tissue inhibitor of metalloproteinases-3 to overcome the weakening effects of the cysteine-rich domains of tumour necrosis factor-α converting enzyme

Lee, Meng-Huee, Dodds, Philippa, Verma, Vandana, Maskos, Klaus, Knauper, Vera ORCID: https://orcid.org/0000-0002-3965-9924 and Murphy, Gillian 2003. Tailoring tissue inhibitor of metalloproteinases-3 to overcome the weakening effects of the cysteine-rich domains of tumour necrosis factor-α converting enzyme. Biochemical Journal 371 (2) , pp. 369-376. 10.1042/bj20021538

Full text not available from this repository.

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 <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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Dentistry
Publisher:	Portland Press
ISSN:	0264-6021
Date of Acceptance:	30 January 2003
Last Modified:	08 Sep 2023 14:00
URI:	https://orca.cardiff.ac.uk/id/eprint/161618

Actions (repository staff only)

Edit Item