biostudies-literatureNguyen DMCanadian Government | Canadian Institutes of Health ResearchCanadian Government | Canadian Institutes of Health Research (CIHR)e2315018121https://www.ebi.ac.uk/biostudies/studies/S-EPMC11047111biostudies-literatureUnknown121(17)Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3^KCTD5) that ubiquitylates Gβγ and reduces Gβγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3^NTD/Gβ₁γ₂ assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5^BTB/CUL3^NTD and KCTD5^CTD/Gβγ moieties of the structure. CRL3^KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gβγ in an E3-E3 superassembly, 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/CUL3^NTD/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.Proceedings of the National Academy of Sciences of the United States of AmericaStructure and dynamics of a pentameric KCTD5/CUL3/Gβγ E3 ubiquitin ligase complex.PMC11047111PJT-159687PJT-191900Petrin DHebert TENguyen DMDevost DZhai APomroy NCBenlekbir SKeszei AFAKuntz DAMazhab-Jafari MTRizk RRubinstein JLPrive GGRath DHJi AXYong DNarayanan NMian MUQfalseStructure and dynamics of a pentameric KCTD5/CUL3/Gβγ E3 ubiquitin ligase complex.Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3^KCTD5) that ubiquitylates Gβγ and reduces Gβγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3^NTD/Gβ₁γ₂ assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5^BTB/CUL3^NTD and KCTD5^CTD/Gβγ moieties of the structure. CRL3^KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gβγ in an E3-E3 superassembly, 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/CUL3^NTD/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.2024-01-01T00:00:00Z2024 Apr2026-07-01T03:08:27.702Z2025-04-07T00:52:20.858ZS-EPMC110471113862594010.1073/pnas.2315018121