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.
Abbreviations used: ADAM, a disintegrin and metalloproteinase; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; EGF, epidermal growth factor; gel-A, gelatinase-A (also known as MMP-2); K26E, etc., mutant bearing an amino acid substitution of Lys26→Glu, etc.; MMP, matrix metalloproteinase; N-TIMP-3, N-terminal-domain form of TIMP-3; OB, oligosaccharide/oligonucleotide binding; PDB, Protein Data Bank; TNF-α, tumour necrosis factor-α; TACE, TNF-α converting enzyme; TACE-cat, the catalytic domain form of TACE; TACE-long, TACE incorporating the catalytic and the cysteine-rich domains; TIMP, tissue inhibitor of metalloproteinases.
Present address: Department of Biology, Biomedical Tissue Research Group, University of York, Heslington, York YO10 5YW, U.K.