Visualizing the chaperone-mediated folding trajectory of the G protein β5 β-propeller by high resolution cryo-EM

Abstract

The cytosolic chaperonin CCT and its co-chaperone phosducin-like protein 1 (PhLP1) play important roles in G protein heterotrimer assembly by folding G protein b subunits (Gb) into b-propeller structures. To understand this process at the molecular level, we have isolated the CCT-Gb 5 -PhLP1 folding intermediate in both the open and closed CCT conformations and determined its structure by high resolution cryo-electron microscopy (cryo-EM). In the open structures, Gb 5 interacts in an unstructured state with the N-and C-termini of the CCT subunits deep inside the folding chamber between the CCT rings. Two copies of PhLP1 bind to the apical domains at the rim of the folding chamber on either end of CCT, allosterically enhancing Gb 5 binding to CCT. In the closed CCT structure, Gb 5 moves from between the CCT rings into one of the folding chambers, suggesting a path for release of Gb 5 from CCT during its ATPase cycle. The other chamber is occupied by one copy of PhLP1, which reaches across to Gb 5 in the opposite folding chamber to stabilize Gb 5 folding. 3D classification and variability analysis of the closed structure captured Gb 5 in progressively folded states that reveal its folding trajectory. CCT initiates folding on Gb 5 blade 4 and folding progresses radially around the b-propeller as CCT makes contacts with blades 3 and 2 in one direction and blades 5 and 6 in the opposite direction. Closing of the b-propeller occurs with the folding of blades 1 and 7 without making additional contacts with CCT. Unexpectedly, CCT interacts exclusively with hydrophilic surface residues of Gb 5 , which leaves the hydrophobic core free to coalesce into its b-sheet structures. These findings provide unprecedented molecular views of CCT-dependent folding of Gb 5 that prepares it to interact with RGS proteins and perform their essential functions in G protein signaling.