The Role of KLHDC2 in Recognizing Diglycine C-end Degron and its Therapeutic Potential

Abstract

Ubiquitin-proteasome system is crucial for controlling essential cellular processes. Eukaryotic cells conjugate the eight kDa ubiquitin polypeptide to a variety of proteins to regulate their functions or turnover. Modification of substrate proteins by ubiquitin is promoted by a three-enzyme cascade consisting of the ubiquitin activating E1 enzyme, ubiquitin conjugating E2 enzyme, and ubiquitin E3 ligase. At the final step of ubiquitin transfer, many ubiquitin E3 ligases recognize specific substrates and promote their poly-ubiquitination, which often results in proteasomal degradation. Among the three known classes of E3s, cullin-RING ubiquitin ligases (CRLs) are the largest family of multi-subunit E3s. With a modular assembly, CRLs are capable of recruiting a myriad of substrates onto a common catalytic scaffold by employing interchangeable substrate receptor subunits. Engagement between a specific substrate and its cognate receptor is frequently mediated by a short linear amino acid sequence in the target protein, commonly known as a degron. Despite years of progress, there is still a major gap in our understanding of degron recognition by CRLs. Recent studies have identified a new class of degrons, which are localized at the C-terminal end of a panel of abnormal polypeptides. These so-called C-end degrons are specifically recognized by a subfamily of CRL2s with previously unknown functions. In my Ph.D. thesis studies, I determined three crystal structures of a novel CRL2 substrate receptor, KLHDC2, in complexes with the diglycine-ending C-end degrons of two early terminated selenoproteins and the N-terminal proteolytic fragment of USP1. KLHDC2 recognizes these C-end degron peptides with a similar coiled conformation and cradles their common C-terminal diglycine motif with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. My results reveal the structural mechanism by which KLHDC2 recognizes the simplest C-end degron and suggest a functional necessity of the E3 to tightly maintain the low abundance of its select substrates. With a virtual screen approach, I have also established KLHDC2 as a potential new platform for targeted protein degradation, which represents a new strategy in therapeutic development.