Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of metalloproteinases (MPs), which are zinc-dependent endopeptidases with key roles in degradation and remodeling of the extracellular matrix (ECM). Dysregulation of MPs is long known for its contribution to several diseases such as cancer, neurological disorders, and cardiovascular diseases. TIMPs, a protein family of four members in humans, show a spectrum of binding affinity and selectivity to different MPs as well as other unique cell signaling binding partners, despite TIMPs having high sequence and structure similarity. TIMPs are comprised of two domains, with the N-terminal domain known as the main inhibitory domain. Understanding the underlying binding mechanism of TIMP-MP binding based on sequence and structure has a great importance in developing the next generation of effective protein-based therapeutics targeting MPs.<br/><br/>We used yeast surface display and protein engineering techniques such as DNA shuffling and directed evolution to identify a minimal inhibitory domain and fragment in the TIMP family. First, we displayed full-length and N-terminal domains of the TIMP family (TIMP-1, -2, -3, -4) on the yeast surface and analyzed binding and expression using flow cytometry. Various TIMPs displayed varied levels of expression and MP binding on the yeast surface, with full-length TIMP-3 having the lowest binding and expression. Additionally, we screened a scrambled TIMP library, made using DNA shuffling within the TIMP family, for expression and MP binding via fluorescent-activated cell sorting (FACS). Interestingly, several scrambled TIMP fragments with sizes as low as 40 amino acids were isolated after rounds of FACS screening, with maintained or improved binding to MMP-3 catalytic domain. The study allowed us to explore any TIMP motifs involved in binding to MP or other cell signaling targets, and hypothetically engineer more functional, tissue-penetrable and selective peptide drug candidates.

Engineering and design of a minimal metalloproteinase inhibitor scaffold

ACS Fall 2021

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