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bioRxiv · 10.1101/2022.08.08.503128

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

Cong, Y.·Liu, C.·Jin, M.·Wang, S.·Han, W.·Zhao, Q.·Wang, Y.·Xu, C.·Diao, L.·Yin, Y.·Peng, C.·Bao, L.
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Abstract

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 human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. Our structural and XL-MS analyses suggested a gradual upward translocation and stabilization of tubulin within the TRiC chamber accompanying TRiC ring closure. Remarkably, in the closed TRiC-tubulin-S3 map resolved to 3.1-[A]-resolution, we captured a near-natively folded tubulin. We found the near-natively folded 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, while the tubulin I domain was found to remain dynamic. Moreover, we also showed the potential role of TRiC C-terminal tails in substrate stabilization and folding. Our study delineates the pathway and molecular mechanism of TRiC-mediated tubulin folding conjugated with TRiC ATPase cycle, and may also inform the design of therapeutic agents targeting TRiC-tubulin interactions.

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Cong, Y., Liu, C., Jin, M., Wang, S., Han, W., Zhao, Q., Wang, Y., Xu, C., Diao, L., Yin, Y., Peng, C., Bao, L.. 2022-08-08. Pathway and mechanism of tubulin folding mediated by TRiC/CCT conjugated with its ATPase cycle revealed by cryo-EM. https://doi.org/10.1101/2022.08.08.503128

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