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Pathway and mechanism of tubulin folding mediated by TRiC/CCT along its ATPase cycle revealed using cryo-EM

Caixuan Liu,M. Jin,Shutian Wang,Wenyu Han,Qiaoyu Zhao,Yifan Wang,Cong Xu,Lei Diao,Yue Yin,Chao Peng,Lan Bao,Yanxing Wang,Y. Cong

Published 2023 in Communications Biology

ABSTRACT

Cryo-EM and XL-MS analyses reveal the conformational landscape of TRiCmediated tubulin folding along TRiC’s ATPase cycle and the interaction sites between tubulin and the closed TRiC chamber. The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of endogenous human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. The open-state TRiC-tubulin-S1 and -S2 maps show extra density corresponding to tubulin in the cis-ring chamber of TRiC. Our structural and XL-MS analyses suggest a gradual upward translocation and stabilization of tubulin within the TRiC chamber accompanying TRiC ring closure. In the closed TRiC-tubulin-S3 map, we capture a near-natively folded tubulin—with the tubulin engaging through its N and C domains mainly with the A and I domains of the CCT3/6/8 subunits through electrostatic and hydrophilic interactions. Moreover, we also show the potential role of TRiC C-terminal tails in substrate stabilization and folding. Our study delineates the pathway and molecular mechanism of TRiC-mediated folding of tubulin along the ATPase cycle of TRiC, and may also inform the design of therapeutic agents targeting TRiC-tubulin interactions.

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