Structural Elucidation of Engineered Tissue Inhibitor of Metalloproteinases‐1 (TIMP‐1) Variants with Improved Binding Affinity toward Matrix Metalloproteinase‐3 (MMP‐3)

Abstract

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.Support or Funding InformationThis work was supported by grants DOD W81XWH‐16‐2‐0030, and NIH R21 CA205471. Diffraction data were measured at the Berkeley National Synchrotron Light Source.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.