Abstract
The TRiC/CCT complex assists in the folding of approximately 10% of cytosolic proteins through an ATP-driven conformational cycle, playing a crucial role in maintaining protein homeostasis. Despite our understanding of ATP-driven TRiC ring closing and substrate folding, the process and mechanisms underlying TRiC ring-opening and substrate release remain largely unexplored. In this study, by determining an ensemble of cryo-EM structures of yeast TRiC in the presence of ADP, including three intermediate transition states, we present a comprehensive picture of the TRiC ring-opening process. During this process, CCT3 detects the loss of γ-phosphate and initiates with the dynamics of its apical protrusion, and expands to the outward leaning of the consecutive CCT6/8/7/5 subunits. This is followed by significant movements of CCT2, CCT4, and especially CCT1 subunits, resulting in the opening of the TRiC rings. We also observed an unforeseen temporary separation between the two rings in the CCT2 side, coordinating the release of the originally locked CCT4 N-terminus, which potentially participates in the ring-opening process. Collectively, our study reveals a stepwise TRiC ring-opening mechanism, provides a comprehensive view of the TRiC conformational landscape, and sheds lights on its subunit specificity in sensing nucleotide status and substrate release. Our findings deepen our understanding of protein folding assisted by TRiC and may inspire new strategies for the diagnosis and treatment of related diseases.