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. Although important aspects of its structural organization and protein interactions have been elucidated, additional questions remain regarding how these features relate to cargo recognition and complex dynamics. 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 BBS1^M390R 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. AlphaFold3-mediated analysis of BBSome interactions with G protein-coupled receptors (GPCRs) led to the identification of contact hotspots on BBS1, BBS4, and BBS5. These predictions were supported by immunoprecipitation and peptide competition assays. The modeling also suggested plausible interfaces between specific BBS proteins and metabolic signaling proteins, including MRAP2 (an MC4R chaperonin), the leptin receptor, and the insulin receptor. These predicted interfaces align with previously reported biochemical associations between BBS proteins and these receptors, supporting the idea that the BBSome regulates trafficking and signaling in 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.