Structure and dynamics of a pentameric KCTD5/Cullin3/Gβγ E3 ubiquitin ligase complex

Abstract

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.

A BTB extension and ion-binding domain contribute to the pentameric structure and TFAP2A binding of KCTD1

“…The BTB and CTD domains are tethered by a helix that may allow functional interplay between the two pentameric rings, as evidenced by E3 ligases such as KCTD5 6,7 . We note that the CTD of KCTD5 displays a relatively 'open' solvent-filled central channel, whereas the channel of KCTD1 is restricted to bind to alternating sodium and iodide ions, the most unusual feature of the structure.…”

“…The structure helps to define a 40-residue insertion (βB-αA-βC-αB) relative to KCTD5 that enables its structural conservation to be recognized (Figures 1C-D). In the pentameric assembly, these structural elements lie on the outermost face of the β-propeller-like fold, where they potentially form a novel interface for binding partners, as observed for the prototypical KCTD5 6,7 .…”

“…Structural studies have been central to their characterization. The prototypical KCTD5 was the first member to have its protein structure elucidated and remains the only family member for which the available structures span both domains [5][6][7] . This protein is observed to act as an off-switch for GPCR signalling through the ubiquitin-mediated degradation of G-protein βγ subunits 6,8,9 .…”

“…This protein is observed to act as an off-switch for GPCR signalling through the ubiquitin-mediated degradation of G-protein βγ subunits 6,8,9 . Its BTB domain displays significant structural conservation with the potassium (K + ) channel tetramerization domain (T1), from which the KCTD family derives its name, but adopts a closed pentameric assembly that binds to cullin3 with 5:5 stoichiometry 5,7 . The associated pentameric CTD of KCTD5 resembles a beta-propeller-like structure that can similarly assemble as a 5:5 complex with Gβγ subunits, further highlighting the importance of oligomerization and cooperative binding for KCTD family function 6,7 .…”

Structural studies of KCTD1 and its disease-causing mutant P20S provide insights into the protein function and misfunction

“…For many years, a full understanding of the functionality of these proteins has been hampered by the paucity of related atomiclevel structural data (12)(13)(14). This scenario has rapidly changed in the last few years through the combination of experimental structural studies (15)(16)(17)(18)(19)(20) and approaches based on machine-learning techniques (21)(22)(23). These studies have provided a plausible global view of the structural features of these proteins and their interaction with some important biological partners.…”