Bardet-Biedl syndrome (BBS) is a rare genetic disease that causes retinal degradation, obesity, kidney dysfunction, polydactyly, and other cilium-related disorders. To date, more than 20 BBS genes, whose mutants cause BBS phenotypes, have been identified, and eight of those (BBS1-2, 4-5, 7-9, and 18) are known to form the BBSome complex. Recent studies have revealed that the BBSome is closely involved in the trafficking of signaling proteins in the primary cilium. Mutations in BBS genes are highly pathogenic because trafficking in the primary cilium is not fully functional when BBS mutations impair assembly of the BBSome. However, the functional links between onset of BBS and BBSome assembly are not well understood. To address this gap in knowledge, we examined the structure of a BBSome assembly intermediate, the BBSome core complex (BBS2, 7, and 9). We employed a combination of chemical crosslinking coupled with mass spectrometry (XL-MS) and electron microscopy (EM) to determine the structure. We applied this structural information to BBS mutations in the core complex to understand how these mutations might cause the disease. These results provide the first structural model of the BBSome core complex and give insight into the molecular basis of Bardet-Biedl syndrome. We have also investigated the mechanism of assembly of the two mTOR kinase complexes (mTORC1 and 2). mTOR is a master regulator of cell metabolism, growth and proliferation. As such, mTOR is a high-value drug target. We investigated the mechanism of assembly of these mTOR complexes and found that the cytosolic chaperonin CCT contributes to mTOR signaling by assisting in the folding of mLST8 and Raptor, components of mTORC1 and mTORC2. To understand the function of CCT in mTOR complex assembly at the molecular level, we have isolated the mLST8-CCT complex and performed a structural analysis using chemical cross-linking couple with mass spectrometry (XL-MS) and cryogenic EM. We found that mLST8 binds CCT deep in its folding cavity, making specific contacts with the CCTα and γ subunits and forming a near-native β-propeller conformation. This information can be used to develop new therapeutics that regulate mTOR activity by controlling mTOR complex assembly.

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G protein signaling depends on the ability of the individual subunits of the G protein heterotrimer to assemble into a functional complex. Formation of the G protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings the Gβ and Gγ subunits together. This system includes the cytosolic chaperonin containing TCP-1 (CCT) and a co-chaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1-Gβ-CCT complexes, were isolated and their structures determined by cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. These structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ β-propeller, disrupting interactions with CCT and releasing a PhLP1-Gβ dimer for assembly with Gγ. We also investigated the role of CCT and PhLP1 in folding and assembling mTOR complexes, which regulate cell growth through phosphorylation. We found that the β-propeller protein mLST8 and one of its binding partners called raptor, which is a large protein in which one domain forms a β-propeller, both bind to CCT. PhLP1 forms a ternary complex with mLST8 and CCT and may play a co-chaperone role. Depletion of PhLP1 or CCT reduces assembly of mTOR complexes in the cell. Collectively, this report reveals diversity in the contributions of CCT to the formation of protein complexes in signaling pathways and presents a molecular mechanism of Gβ folding by CCT and PhLP1.