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

Meng Huee Lee, Philippa Dodds, Vandana Verma, Klaus Maskos, Vera Knäuper, Gillian Murphy*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

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 proinflammatory 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 <0.1 nM, these mutants were dramatically better than the wild-type N-TIMP-3 [Ki (app) 1.7 nM]. 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 re-orientation 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.

Original languageEnglish
Pages (from-to)369-376
Number of pages8
JournalBiochemical Journal
Volume371
Issue number2
DOIs
Publication statusPublished - 15 Apr 2003
Externally publishedYes

Keywords

  • Binding affinity
  • Conformation tuning
  • Protein engineering

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