Mechanisms of chaperone-mediated folding of beta-propeller proteins essential for vision. - PROJECT SUMMARY The cytosolic chaperonin CCT is a large protein complex that plays an indispensable role in maintaining the cellular proteome by assisting in the folding of numerous proteins with complex tertiary structures and unfavorable folding trajectories. Proper CCT function is vital to human vision as evidenced by the fact that inactivating mutations in CCT cause Leber Congenital Amaurosis (LCA). CCT contributes to the visual process by folding the cytoskeletal proteins actin and tubulin as well as other proteins with b-propeller folds that have essential functions in vison. These include the G protein b1 (Gb1) subunit of the visual G protein transducin, the G protein b5 (Gb5) subunit of the regulator of G protein signaling 9 (RGS9) dimer, and the BBS2 and BBS7 subunits of the Bardet-Biedl syndrome (BBS) ciliary transport complex, the BBSome. Despite the importance of CCT in maintaining the proteome, we know very little at the molecular level about how CCT assists in the folding of these b-propeller proteins and how mutations disrupt folding and cause disease. To address this gap in knowledge, we propose to determine the structures of human Gb1 and Gb5 and their disease-causing mutants. Structures of Gb5 bound to CCT and its co-chaperone PhLP1 show progressive step-by-step formation of the Gb5 b-propeller that reveals its folding trajectory. Unraveling how CCT influences the folding trajectory of a b-propeller protein represents a breakthrough in understanding chaperone-mediated protein folding. Moreover, applying these same techniques to misfolding and disease-causing mutants of Gb1 and Gb5 will show how the mutations disrupt their folding trajectories. Finally, we propose to employ our biochemical and high resolution cryo-EM expertise to understanding biogenesis of the BBSome complex. A key step in BBSome assembly is the formation of the BBS2-BBS7 dimer, which requires both CCT and three chaperonin- like (CL-BBS) proteins BBS6, BBS10 and BBS12 to come together. Despite the 18 years since CL-BBS protein discovery and the predominant role their mutations play in causing BBS, the molecular mechanism by which the CL-BBS proteins and CCT assist in BBS2 and BBS7 folding and BBS2-BBS7 dimer formation is unknown. The proposed studies will fill this gap in knowledge and will deepen understanding of the molecular defects caused by mutations in Gb subunits, BBS7 and CL-BBS proteins. The structural work will establish a foundation for targeted, structure-based drug design to create new therapies for the retinopathies, neuropathies and ciliopathies caused by these mutations.
