Cryo-EM visualizes substrate channeling and E2 multisite phosphorylation configuring ubiquitin ligation by GID/CTLH E3

Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including 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, cryo-EM, biochemistry, and cell biology reveal exquisitely specific GID/CTLH-Ubc8/UBE2H pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 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 illustrate phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, immobilizes the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.