Structural basis for the ubiquitination of G protein βγ subunits by KCTD5/Cullin3 E3 ligase

Area of Science:

• Molecular Biology
• Structural Biology
• Cell Signaling

Background:

• G protein-coupled receptor (GPCR) signaling requires precise regulation to prevent detrimental overstimulation.
• Gβγ signaling, a component of GPCR pathways, is negatively controlled by the Cullin3 (Cul3)-dependent E3 ligase KCTD5.
• KCTD5 targets free Gβγ for ubiquitination and subsequent degradation.

Purpose of the Study:

• To elucidate the structural basis of KCTD5-mediated Gβγ regulation.
• To investigate the mechanism of substrate recognition and ubiquitination by the KCTD5/Cul3 E3 ligase complex.
• To understand the role of KCTD5 in fine-tuning GPCR signaling pathways.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structures of the KCTD5-Gβγ fusion complex and the KCTD7-Cul3 complex.
• Structural analysis focused on the pentameric assembly of KCTD5 and its interaction with Gβγ subunits.
• Functional assays assessed the impact of KCTD5-mediated ubiquitination on Gβγ and cyclic adenosine 3´,5´-monophosphate (cAMP) signaling.

Main Results:

• The cryo-EM structures revealed a pentameric KCTD5 engaging symmetrically with five Gβγ subunits via its C-terminal domain.
• The unique pentameric E3 ligase assembly facilitates proximity between the ubiquitin-conjugating enzyme (E2) and Gβγ modification sites.
• Simultaneous ubiquitination of multiple Gβγ subunits by KCTD5 was observed, impacting GPCR-mediated cAMP signaling.

Conclusions:

• KCTD5 functions as a pentameric E3 ligase, uniquely recognizing and ubiquitylating multiple Gβγ subunits.
• This mechanism provides precise control over Gβγ signaling and fine-tunes GPCR-mediated cAMP responses.
• The KCTD family emerges as significant regulators of GPCR signaling, with KCTD5 offering insights into unusual E3 ligase assembly and substrate recognition.