Reactive site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: Biological and structural implications.
Résumé
We have previously reported that reactive site mutants of the N-terminal inhibitory domain of TIMP-3 (N-TIMP-3) modified at the N-terminus, selectively inhibited ADAM17 (A Disintegrin and Metalloprotease) over the MMPs (Matrix Metalloproteases). The primary aggrecanases, ADAMTS-4 and ADAMTS-5 (ADAM with Thrombospondin motifs) are ADAM17-related metalloproteases which are similarly inhibited by TIMP-3, but are poorly inhibited by other TIMPs. Using a newly developed recombinant protein substrate based on the interglobular domain (IGD) of aggrecan, gst-IGD-flag, these reactive site mutants were found to similarly inhibit ADAMTS-4 and ADAMTS-5. Further mutations of N-TIMP-3 indicated that up to two extra Ala residues can be attached to the N-terminus before the Ki(app) for ADAMTS-4 and ADAMTS-5 increased to over 100 nM. No other residues tested at the [- 1] position produced inhibitors as potent as the Ala mutant. The mutants N-TIMP-3(T2G), [- 1A]N-TIMP-3 and [-2A]N-TIMP-3 were effective inhibitors of aggrecan degradation but not of collagen degradation in both interleukin-1α (IL-1α)-stimulated porcine articular cartilage explants and IL-1α with oncostatin M-stimulated human cartilage explants. Molecular modelling studies indicated that the [-1A]N-TIMP-3 mutant has additional stabilising interactions with the catalytic domains of ADAM17, ADAMTS-4 and ADAMTS-5 that are absent in complexes with MMPs. These observations suggest that further mutation of the residues of N-TIMP-3 which make unique contacts with these metalloproteinases may allow discrimination between them.
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