Heterotrimeric G proteins can be regulated by post-translational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain (CTD), engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gβγ and reduces Gβγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gβ1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gβγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gβγ in an E3-E3 super-assembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gβ and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD Gβγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.

Relion Multi-body cryo-EM maps and PDB models of states A, B, C, and D of a KCTD5/Cul3(NTD)/Gbeta1gamma2 complex. Each zipped file contains a multimodel cif file consisting of 10 structures and the corresponding 10 maps into which the structures were fitted. Movie file KCTD5_Cul3_Gbg_states_ABCD_with_Multibody.mp4 was generated from the 40 models and is also included with the main paper.

{"references": ["Nguyen, DM et al. (2023). Structure and dynamics of a pentameric KCTD5/Cullin3/G\u03b2\u03b3 E3 ubiquitin ligase complex. bioRxiv 10.1101/2023.09.20.558662"]}