Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, National Center for Macromolecular Imaging, Baylor College of Medicine, Houston, TX 77030, USA.
The EMBO Journal (Impact Factor: 10.43). 11/2011; 31(3):720-30. DOI: 10.1038/emboj.2011.366
Source: PubMed


The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction. These structures reveal the intra- and inter-ring subunit interaction pattern changes during the ATPase cycle. In the apo state, the subunit arrangement in each ring is highly asymmetric, whereas all nucleotide-containing states tend to be more symmetrical. We identify and structurally characterize an one-ring closed intermediate induced by ATP hydrolysis wherein the closed TRiC ring exhibits an observable chamber expansion. This likely represents the physiological substrate folding state. Our structural results suggest mechanisms for inter-ring-negative cooperativity, intra-ring-positive cooperativity, and protein-folding chamber closure of TRiC. Intriguingly, these mechanisms are different from other group I and II chaperonins despite their similar architecture.

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    • "CCT is a cylindrical, 16-subunit toroid composed of eight distinct subunits (CCTa–CCTh) that form two eight subunit, back-to-back rings, each containing a folding ''cage'' for CPs (Yébenes et al., 2011; Leitner et al., 2012). Sequestration of CPs by CCT in a closed conformation and CP release require ATP binding, hydrolysis, and associated intra-and inter-ring allosteric signaling (Yébenes et al., 2011; Leitner et al., 2012; Cong et al., 2012). "
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    • "We applied a consecutive multiple-model refinement strategy, previously used to successfully analyze images of chaperonins with mixed conformations and compositions (Chen et al., 2006, 2008; Cong et al., 2012). The first round of processing of 71,200 particle images yielded three subpopulations of images that resulted in free GroEL tetradecamer (no GroES bound), bullet-shaped GroEL-GroES complexes (with GroES bound to only one end of the GroEL tetradecamer) and football-shaped GroEL-GroES 2 complexes (with GroES bound to both ends of the GroEL tetradecamer). "
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    • "Accordingly, we refined the structure without imposing symmetry while sorting the data set through the use of codimensional principal component analysis (CD-PCA) (Penczek et al., 2011) followed by multireference refinement and subsequently noticed that at least one of the protein chains composing the homohexamer was in a distinct conformation. It should also be noted that studies on the chaperonin TRiC (Cong et al., 2012) and GroEL (Clare et al., 2012) were similarly able to identify conformational changes within structures previously assumed to display higher order symmetry. This symmetry-free approach yielded three distinguishable and interpretable EM maps containing 16,830 particle projections (Figures 1D–1F) from the final data set of 22,730 projections . "
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