Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 280, Issue 16, Pages 15967-15975Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M500897200
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Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases, the ADAMs ( a disintegrin and metalloproteinase) and the ADAM-TS ( ADAM with thrombospondin repeats) proteinases. There are four mammalian TIMPs (TIMP-1 to -4), and each TIMP has its own profile of metalloproteinase inhibition. TIMP-4 is the latest member of the TIMPs to be cloned, and it has never been reported to be active against the tumor necrosis factor-alpha-converting enzyme ( TACE, ADAM-17). Here we examined the inhibitory properties of the full-length and the N-terminal domain form of TIMP-4 ( N-TIMP-4) with TACE and showed that N- TIMP-4 is a far superior inhibitor than its full-length counterpart. Although full-length TIMP-4 displayed negligible activity against TACE, N- TIMP-4 is a slow tight-binding inhibitor with low nanomolar binding affinity. Our findings suggested that the C-terminal subdomains of the TIMPs have a significant impact over their activities with the ADAMs. To elucidate further the molecular basis that underpins TIMP/TACE interactions, we sculpted N-TIMP-4 with the surface residues of TIMP-3, the only native TIMP inhibitor of the enzyme. Transplantation of only three residues, Pro-Phe-Gly, onto the AB-loop of N- TIMP-4 resulted in a 10-fold enhancement in binding affinity; the K-i values of the resultant mutant were almost comparable with that of TIMP-3. Further mutation at the EF-loop supported our earlier findings on the preference of TACE for leucine at this locus. Drawing together our previous experience in TACE-targeted mutagenesis by using TIMP-1 and -2 scaffolds, we have finally resolved the mystery of the selective sensitivity of TACE to TIMP-3.
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