Engineering a U-box of E3 ligase E4B through yeast surface display-based functional screening generates a variant with enhanced ubiquitin ligase activity
Ubiquitination is the covalent attachment of ubiquitin (Ub) to substrate proteins and regulates several cellular processes, including protein degradation. Ub ligases (E3s) bring a Ub-conjugated enzyme E2 (E2–Ub) and the target protein closer to enable ubiquitination. In this study, we engineered a U-box domain of human U-box-type E3 E4B (E4BU) to enhance its function as a Ub ligase by accelerating the rate of Ub transfer directly from Ub-loaded human E2 UbcH5b (E2(UbcH5b)–Ub) to the proximal substrate. We developed a functional screening system for the E4BU library using a yeast surface display system combined with fluorescence-activated cell sorting (FACS) to isolate functionally improved variants. This phenotypic screening system yielded an E4BU variant, E4BU(#8), which exhibited an approximately 4-fold greater Ub ligase activity rate in the yeast displayed form than that of the E4BU wild-type. When E4BU(#8) was fused to a green fluorescent protein (GFP)-specific nanobody, the fusion protein polyubiquitinated GFP in proportion to the concentration and incubation time, with an approximately 3-fold faster Ub ligase activity rate than the previously isolated E4BU(NT) variant. Importantly, the engineered E4BU(#8) retained endogenous Lys48-linked polyubiquitination activity, which is essential for substrate degradation by the 26S proteasome. Our results indicated that E4BU(#8), which binds to and allosterically stimulates E2(UbcH5b)–Ub to enhance Ub transferase activity to a substrate, may be valuable in designing biological molecules for targeted protein degradation.
This work was supported by the National Research Foundation of Korea (NRF) [grant number 2021R1A2C2003362 to YSK] and the Priority Research Centers Program [grant number 2019R1A6A1A11051471 to YSK)], which is funded by the Korean government ( MSIT ). The sponsors had no role in the study design; collection, analysis, and interpretation of the data; writing of the report; and decision to submit the manuscript for publication.