No abstract

The cytosolic chaperonin CCT and its co‐chaperone phosducin‐like protein 1 (PhLP1) play important roles in G protein complex assembly by folding G protein β subunits (Gβ) into β‐propeller structures. To understand this process at the molecular level, we have isolated the CCT‐Gβ5‐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, Gβ5 interact with the N‐ and C‐termini of the CCT subunits deep inside the folding chamber between the CCT rings in a closed β‐propeller conformation. PhLP1 allosterically enhances Gβ5 binding to CCT by interacting with the CCT apical domains at the rim of the folding chamber without contacting Gβ5 directly. In the closed CCT structures, Gβ5 move from between the CCT rings into one of the folding chambers, suggesting a path for release of Gβ5 from CCT during its ATPase cycle. These findings provide a molecular explanation for CCT‐dependent folding and release of Gβ5 to interact with RGS proteins and perform their essential functions in G protein signaling.

No abstract

The cytosolic Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with β-propeller domains. Here, we determined structures of CCT in complex with its accessory co-chaperone, phosducin-like protein 1 (PhLP1), in the process of folding Gβ5, a component of Regulator of G protein Signaling (RGS) complexes. Cryo-EM and image processing revealed an ensemble of distinct snapshots that represent the folding trajectory of Gβ5 from an unfolded molten globule to a fully folded β-propeller. These structures reveal the mechanism by which CCT directs Gβ5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual β-sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT directs folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.

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.