Disease-causing mutations in the G protein β5 β-propeller disrupt its chaperonin-mediated folding trajectory

Abstract

The Chaperonin Containing Tailless polypeptide 1 (CCT or TRiC) is an essential cytosolic chaperone that folds multiple protein substrates, including many with {beta}-propeller folds. One {beta}-propeller substrate is the G protein {beta}5 subunit (G{beta}5) of Regulator of G protein Signaling (RGS) complexes that determine the duration of G protein signals in neurons. In recent work, we used cryo-electron microscopy (cryo-EM) to visualize the complete CCT-mediated folding trajectory for G{beta}5, from an initiating electrostatic interaction of a single {beta}-strand in G{beta}5 with the CCT5 subunit to a completely folded {beta}-propeller structure. Here, we employed biochemistry and cryo-EM to determine key interactions with CCT that initiate G{beta}5 folding and how missense mutations in G{beta}5 that cause severe neurological diseases alter the G{beta}5 folding trajectory and lead to incompletely folded, trapped intermediates. These findings highlight how CCT recognizes folding substrates, how defects in chaperonin-mediated folding contribute to disease, and how strategies might be designed to stabilize misfolded proteins to restore function. SignificanceElectrostatic interactions between the CCT chaperonin and its protein substrates initiate the folding process. Using cryo-EM structure determinations, we found a striking specificity for these interactions that allow CCT-mediated G{beta}5 folding. Moreover, certain missense mutations in G{beta}5 lead to misfolding and are associated with neurological disorders. We tracked how these mutations disrupt the normal folding of G{beta}5 by CCT. Although mutant G{beta}5 still binds the complex, folding stalls mid-process, leaving the protein trapped in partially folded, non-functional states. These defects arise from disrupted packing of the G{beta}5 core that interferes with formation of the native structure. Our findings reveal a molecular basis for G{beta}5 misfolding in disease and suggest pharmacological chaperones that stabilize the folded state might restore proper function.