Reporting in Cell, Gestaut et al. reconstituted the tubulin folding pathway in vitro using purified human proteins. They monitored tubulin folding via a combination of biochemical and structural methods. The authors observed that prefoldin, a jellyfish-shaped protein, interacted with tubulin via its ‘tentacles’, forming a highly dynamic ensemble, enclosing the unfolded protein in a cage, to retain its unfolded state. They saw that binding of prefoldin induced opening of TRiC and release of the substrate into its chamber. The authors determined the presence of a third, inter-ring chamber, where the unstructured tails of CCT TRiC subunits maintained an unfolded substrate before lid closure. The cryo-EM structures showed that the addition of ATP led to closure and translocation of tubulin to one of the apical chambers. Three-dimensional classification of cryo-EM data revealed four distinct progressively folded tubulin states, which enabled the authors to describe the tubulin folding pathway. They propose that TRiC interacts with folding intermediates through CCT-subunit-specific contacts, to avoid the formation of an energetically trapped misfolded state and to facilitate reaching the native topology. This is thought to be crucial for aggregation-prone β-sheet-rich proteins such as tubulin, and offers an alternative folding mechanism for other chaperones, such as GroEL-ES, which release the substrate into their chamber.
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