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bioRxiv · 10.1101/2022.03.25.483853

Structural visualization of the tubulin folding pathway directed by eukaryotic chaperonin TRiC

Gestaut, D. R.·Zhao, Y.·Park, J.·Ma, B.·Leitner, A.·Collier, M.·Pintilie, G.·Roh, S. H.·Chiu, W.·Frydman, J.
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Abstract

SUMMARYThe ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of {beta}-tubulin using human Prefoldin and TRiC. We find unstructured {beta}-tubulin is delivered by Prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. CryoEM resolves four near-atomic resolution structures containing progressively folded {beta}-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N-domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Disordered CCT C-termini within the chamber promote subsequent folding of tubulin Core and Middle domains and GTP-binding. Thus, TRiCs chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

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Gestaut, D. R., Zhao, Y., Park, J., Ma, B., Leitner, A., Collier, M., Pintilie, G., Roh, S. H., Chiu, W., Frydman, J.. 2022-03-26. Structural visualization of the tubulin folding pathway directed by eukaryotic chaperonin TRiC. https://doi.org/10.1101/2022.03.25.483853

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