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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Available from: Judith Frydman, Sep 30, 2015
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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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    ABSTRACT: Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT-CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT-CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lönnberg) (habitat/body T = -1.9 to +2°C), and of the cow (body T = 37°C). We examined the temperature dependence of the binding of denatured CPs (β-actin, β-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between -4°C and 20°C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2°C. The ATPase activity of apo-CCT from G. gibberifrons at 4°C was ∼2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20°C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits.
    Biology Open 03/2014; 3(4). DOI:10.1242/bio.20147427 · 2.42 Impact Factor
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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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    ABSTRACT: The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.
    Cell 06/2013; 153(6):1354-65. DOI:10.1016/j.cell.2013.04.052 · 32.24 Impact Factor
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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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