Differential higher-order superassembly of HECT-type UBE3 ligases controlled by calcium signals

The yeast E4 ligase Hul5 transiently associates with the proteasome during heat shock and proteotoxic stress to maintain cytosolic protein quality control. Two of its human orthologs, UBE3B and UBE3C, have specialized to safeguard mitochondria and inherit Hul5 function, respectively. We determined cryo-electron microscopy structures of full-length human UBE3B and UBE3C in complex with calmodulin at 2.9-3.5 [A] in calcium-free and calcium-saturated conditions. Calmodulin acts as an inter-protomer molecular glue clamping UBE3B into ring-shaped anti-parallel homodimers or asymmetric trimers, and remodels UBE3C into a conformation competent for proteasome association. Calcium binding to calmodulin promotes disassembly of these higher-order complexes, toggling UBE3B/C from E4 to E3 activity. These findings reveal how calmodulin regulates higher-order architectures of E3/E4 ligases to rewire the ubiquitin-proteasome system via calcium signaling.