Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes known for contributions to diseases including cancer and fibrosis. The TIMP N-terminal domain, regarded as the “inhibitory domain”, has been the center of attention both for understanding natural regulation of MMPs by TIMPs, and for developing protein-based MMP inhibitors for therapeutic applications. The role of the TIMP C-terminal domain in MMP inhibition has not been well studied. I have used directed evolution via yeast surface display for protein engineering of full-length human TIMP-1 toward development of MMP-3-targeted ultra-binders. TIMP-1 variants with up to 10-fold improvement in MMP-3 binding (compared to WT-TIMP-1) were isolated after six rounds of library screening using fluorescence-activated cell sorting (FACS). Analysis of individual and paired mutations from the selected TIMP-1 variants revealed a cooperative effect between distant mutations on the N- and C-terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP-3. I have co-crystallized two TIMP-1 variants containing single and composite mutations in complex with the catalytic domain of MMP-3 and solved the X-ray crystal structures of these complexes. The protein structures reveal substantial conformational changes in the TIMP-1 binding interfaces near the two cooperative mutations. Affinity appears to be strengthened by tighter cinching of a reciprocal tyrosine clasp formed between the N-terminal loop of TIMP-1 and the proximal MMP3 interface, and adoption of an alpha-helical conformation by a segment of the TIMP-1 C-terminal domain, resulting in stabilized interactions between the two TIMP-1 domains and improved hydrophobic interactions between TIMP-1 and MMP-3. Our protein engineering and structural studies provide novel insight into the cooperative function of N- and C-terminal TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our results further point to new approaches for design of TIMP-based protein therapeutics with improved affinity and selectivity.

Structural elucidation of engineered tissue inhibitor of metalloproteinase-1 (TIMP-1) variants with improved binding affinity toward matrix metalloproteinase-3 (MMP-3)

2019 Spring National Meeting

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