Reactive-site mutants of N-TIMP-3 that selectively inhibit ADAMTS-4 and ADAMTS-5: biological and structural implications.
We have reported previously that reactive-site mutants of N-TIMP-3 [N-terminal inhibitory domain of TIMP-3 (tissue inhibitor of metalloproteinases 3)] modified at the N-terminus, selectively inhibited ADAM17 (a disintegrin and metalloproteinase 17) over the MMPs (matrix metalloproteinases). The primary aggrecanases ADAMTS (ADAM with thrombospondin motifs) -4 and -5 are ADAM17-related metalloproteinases which are similarly inhibited by TIMP-3, but are poorly inhibited by other TIMPs. Using a newly developed recombinant protein substrate based on the IGD (interglobular domain) 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 alanine 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 alanine 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 IL-1α (interleukin-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 stabilizing interactions with the catalytic domains of ADAM17, ADAMTS-4 and ADAMTS-5 that are absent from 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.
Lim
Ngee H
NH
Kennedy Institute of Rheumatology Division, Imperial College London, 65 Aspenlea Road, London W6 8LH, UK.
Kashiwagi
Masahide
M
Visse
Robert
R
Jones
Jonathan
J
Enghild
Jan J
JJ
Brew
Keith
K
Nagase
Hideaki
H
D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D000071121:ADAMTS4 Protein; D053674:Aggrecans / Q000378:metabolism; D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism; D020134:Catalytic Domain; D002478:Cells, Cultured; D006801:Humans; D009154:Mutation; D011348:Procollagen N-Endopeptidase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D013552:Swine; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D053674:Aggrecans; D019717:Tissue Inhibitor of Metalloproteinase-3; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human