Multisite phosphorylation dictates selective E2-E3 pairing as revealed by Ubc8/UBE2H-GID/CTLH assemblies

• Published in: Molecular cell Vol. 84; no. 2; pp. 293 - 308.e14
• Main Authors: Chrustowicz, Jakub, Sherpa, Dawafuti, Li, Jerry, Langlois, Christine R., Papadopoulou, Eleftheria C., Vu, D. Tung, Hehl, Laura A., Karayel, Özge, Beier, Viola, von Gronau, Susanne, Müller, Judith, Prabu, J. Rajan, Mann, Matthias, Kleiger, Gary, Alpi, Arno F., Schulman, Brenda A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.01.2024
• Subjects: cell biology; CK2; cryo-electron microscopy; cryo-EM; Cryoelectron Microscopy; CTLH complex; dephosphorylation; E2 ubiquitin conjugating enzyme; E3 ubiquitin ligase; evolution; GID complex; human development; Humans; Phosphorylation; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Ubc8; UBE2H; ubiquitin; Ubiquitin-Conjugating Enzymes - metabolism; ubiquitin-protein ligase; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; yeasts; UBE2H; E2 ubiquitin conjugating enzyme; Ubc8; E3 ubiquitin ligase; GID complex; CK2; cryo-EM; phosphorylation; ubiquitin; CTLH complex
• ISSN: 1097-2765; 1097-4164; 1097-4164
• DOI: 10.1016/j.molcel.2023.11.027

Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryoelectron microscopy (cryo-EM), biochemistry, and cell biology reveal this exquisitely specific E3-E2 pairing through an unconventional catalytic assembly and auxiliary interactions 70–100 Å away, mediated by E2 multisite phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data show that phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, rigidifies the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites. [Display omitted] •Multisite phosphorylation of an E2 enzyme mediates E3 binding in vitro and in vivo•Multisite phosphorylation determines dedicated Ubc8-GID and UBE2H-CTLH E2-E3 pairing•Phosphorylated E2 C termini engage evolutionarily correlated E3 basic patches•Flexing oligomeric E3 channels substrate to multiple rigidly placed E2 active sites Chrustowicz, Sherpa, et al. decipher evolutionarily conserved determinants of an exquisitely specific E3-E2 partnership ubiquitylating globular substrates. Cryo-EM visualizes a flexing GID E3 superassembly channeling its substrate between two ubiquitylation active sites. The catalytic architecture depends on multivalent Ubc8/UBE2H-GID/CTLH E3 contacts, including between multiphosphorylated E2 extensions and complementary E3 basic patches.