Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

被引:54
作者
Hamze, Asmaa B.
Wei, Shuo
Bahudhanapati, Harinath
Kota, Smitha
Acharya, K. Ravi
Brew, Keith
机构
[1] Florida Atlantic Univ, Coll Biomed Sci, Dept Biomed Sci, Boca Raton, FL 33431 USA
[2] Univ Bath, Dept Biol & Biochem, Bath BA2 7AY, Avon, England
关键词
matrix metalloproteinase; metalloproteinase inhibition; protein engineering; inhibitory specificity; membrane-type matrix metalloproteinase; interaction interface;
D O I
10.1110/ps.072978507
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The tissue inhibitors of metalloproteinases ( TIMPs) are endogenous inhibitors of the matrix metalloproteinases ( MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (> 3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1( AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and > 40 mu M for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.
引用
收藏
页码:1905 / 1913
页数:9
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