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Design, cyclization, and optimization of MMP13–TIMP1 interaction-derived self-inhibitory peptides against chondrocyte senescence in osteoarthritis
- Zhang, Wei, Zhang, Chi, Luo, Congfeng, Zhan, Yulin, Zhong, Biao
- International journal of biological macromolecules 2019 v.121 pp. 921-929
- active sites, bioinformatics, crystal structure, cyclic peptides, disulfide bonds, entropy, enzyme inhibition, enzyme inhibitors, metalloproteinases, osteoarthritis
- The matrix metallopeptidase 13 (MMP13) is a central regulator of chondrocyte senescence that contributes to the development and progression of osteoarthritis (OA). In the present study, the native inhibitory structure of MMP13 in complex with its natural cognate inhibitor, the tissue inhibitor of metalloproteinases 1 (TIMP1), was modeled at atomic level using a grafting-based structural bioinformatics method with existing crystal structures. The modeled complex structure was then examined in detail, from which a TIMP1 inhibitory site that directly inserts into the active site of MMP13 enzyme was identified. The inhibitory site contains a coiled inhibitory loop (ILP) and a stretched N-terminal tail (NTT); they are highly structured in the intact MMP13–TIMP1 complex interface, but exhibit a large flexibility and intrinsic disorder when split from the interface context. In vitro binding assays demonstrated that the isolated ILP and NTT peptides cannot effectively rebind at the MMP13 active site (Kd > ~100 μM or = n.d.), although they have all key interacting residues in the enzyme inhibition. In silico simulations revealed that splitting of the peptide segments from TIMP1 inhibitory site does not influence the direct intermolecular interaction between MMP13 and the peptides substantially; instead, the large conformational flexibility of these isolated peptides in absence of interface context is primarily responsible for the affinity impairment, which would incur a considerable entropy penalty upon the peptide binding to MMP13. An extended version of ILP peptide, namely eILP (63TPAMESVCGY72), was redesigned with a rational strategy to derive a number of its cyclized counterparts by introducing a disulfide bridge across the peptide two-termini; the redesign reduces the peptide flexibility in free state and constrains the peptide pre-folding to a native-like conformation, which would help the peptide binding with minimized entropy penalty. Binding assays substantiated that the affinity Kd values of four designed cyclic peptides (▪, ▪, ▪ and ▪) were improved to 23, 67, 42 and 18 μM, respectively, from the 96 μM of linear eILP peptide.