We previously reported that full-length tissue inhibitor of metalloproteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-α (TNF-α)-converting enzyme (TACE). Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition. Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature. The activities of these mutants were examined by measuring their binding affinities (Kappi) and association rates (kon) against TACE. Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants. On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE. In fact, the binding affinities of several mutants were less than 60pM, beyond the sensitivity limits of fluorimetric assays. Further studies on cell-based processing of pro-TNF-α demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-α. Furthermore, the Ser-4Met mutant was also significantly more active (P<0.05) than the wild-type N-TIMP-3 in its cellular inhibition. Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF-α shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.
- binding affinity
- N-terminal domain of TIMP-3
↵2 Present address: Department of Biology, Bone and Joint Pathology, University of York, York YO10 5DD, U.K.
↵3 Present address: GlaxoSmithKline, Stevenage, Herts. SG1 2NY, U.K.
Abbreviations used: TIMP, tissue inhibitor of metalloproteinase; N-TIMP-3, N-terminal domain of TIMP-3; TNF-α, tissue necrosis factor-α; TACE, TNF-α-converting enzyme; gel-A, gelatinase-A; ADAM, a disintegrin and a metalloproteinase domain; OB, oligosaccharide/oligonucleotide-binding; MMP, matrix metalloproteinase; GdmCl, guanidinium chloride; FAB fluorescence assay buffer.
- The Biochemical Society, London ©2002