Engineering a U-box of E3 ligase E4B through yeast surface display-based functional screening generates a variant with enhanced ubiquitin ligase activity.
Biochemical and biophysical research communications(2022)
摘要
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.
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