Harnessing AlphaFold3 to Elucidate BBSome Structure and Protein Partners

The BBSome, an eight-protein complex implicated in Bardet-Biedl syndrome (BBS), plays a crucial role in ciliary function, yet its structural organization and protein interactions remain poorly understood. Using AlphaFold3, we generated a structural model closely matching recent cryo-EM data (Cα RMSD: 1.203 Å). Interface residue analysis of the model identified BBSome proteins BBS1 and BBS9 as central interaction hubs (most interface residues between two proteins), with BBS2 and BBS7 showing the most polar contacts. The common BBS1M390R pathogenic mutation, known to cause BBS, was predicted to destabilize the complex. BBS4 was also found to interact stably with pericentriolar material 1, suggesting a role in centriolar satellite localization. We next used AlphaFold3 to examine BBSome interactions with G protein-coupled receptors (GPCRs) which led to the identification of contact hotspots on BBS1, BBS4, and BBS5. These predictions were supported by immunoprecipitation and peptide competition assays. Additionally, AlphaFold3 predicted direct interactions between BBSome subunits and key metabolic proteins and receptors, including MRAP2 (an MC4R chaperone), the leptin receptor (BBS1), and the insulin receptor (BBS17) which validate the previously reported physical associations between BBSome components and these receptors, reinforcing the role of the BBSome in trafficking and signaling regulation of metabolic pathways. Together, these findings provide new insights into BBSome structure and receptor interactions, offering a predictive framework to explore its role in ciliary trafficking and human disease.