Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin

• Published in: Molecular cell 2015-04, Vol.58 (2), p.297-310
• Main Authors: Buetow, Lori, Gabrielsen, Mads, Anthony, Nahoum G., Dou, Hao, Patel, Amrita, Aitkenhead, Hazel, Sibbet, Gary J., Smith, Brian O., Huang, Danny T.
• Format: Article
• Language: English
• Subjects: Crystallography, X-Ray; Models, Molecular; Molecular Dynamics Simulation; Molecular Sequence Data; Ubiquitin - metabolism; Ubiquitin-Conjugating Enzymes - chemistry; Ubiquitin-Conjugating Enzymes - metabolism; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - metabolism; Zinc Fingers
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2015.02.017

RING ubiquitin ligases (E3) recruit ubiquitin-conjugate enzymes (E2) charged with ubiquitin (Ub) to catalyze ubiquitination. Non-covalent Ub binding to the backside of certain E2s promotes processive polyUb formation, but the mechanism remains elusive. Here, we show that backside bound Ub (UbB) enhances both RING-independent and RING-dependent UbcH5B-catalyzed donor Ub (UbD) transfer, but with a more prominent effect in RING-dependent transfer. UbB enhances RING E3s’ affinities for UbcH5B–Ub, and RING E3-UbcH5B–Ub complex improves UbB’s affinity for UbcH5B. A comparison of the crystal structures of a RING E3, RNF38, bound to UbcH5B–Ub in the absence and presence of UbB, together with molecular dynamics simulation and biochemical analyses, suggests UbB restricts the flexibility of UbcH5B’s α1 and α1β1 loop. UbB supports E3 function by stabilizing the RING E3-UbcH5B–Ub complex, thereby improving the catalytic efficiency of Ub transfer. Thus, UbB serves as an allosteric activator of RING E3-mediated Ub transfer. [Display omitted] •UbB activates RING E3-UbcH5B∼Ub complex for UbD transfer•Free Ub, E2∼Ub, and monoUb and polyUb chains on E3 and substrate act as UbB sources•UbB enhances RING E3-UbcH5B–Ub affinity, and RING E3-UbcH5B–Ub improves UbB affinity•Structure-based insights into UbB stimulatory mechanism Buetow et al. show that non-covalent ubiquitin binding to the backside of E2 stimulates RING E3-catalyzed initial ubiquitin transfer and poly-ubiquitin chain formation. Crystal structures and biochemical analyses reveal that backside bound ubiquitin stabilizes RING E3-E2∼ubiquitin complex for catalysis.