Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber

Douglas, Nicholai R.; Reißmann, Stefanie; Zhang, Junjie; Chen, Bin; Jakana, Joanita; Kumar, Ramya; Chiu, Wah; Frydman, Judith

doi:10.1016/j.cell.2010.12.017

Cited by 98 publications

(91 citation statements)

References 37 publications

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“…The mechanism and high-resolution structure of group II chaperonins has been elucidated using the archaeal chaperonin of Methanococcus maripauldis, Mm-Cpn (Douglas et al 2011;Pereira et al 2012Pereira et al , 2010Zhang et al 2010). Due to the increased complexity of TRiC, much of the structural and biochemical research on group II chaperonins has been carried out with less complex archaeal chaperonins, such as Mm-Cpn.…”

Section: Introductionmentioning

confidence: 99%

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Knee

Sergeeva

King

2013

Cell Stress and Chaperones

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Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1-8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing offpathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells.

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Section: Introductionmentioning

confidence: 99%

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Knee

Sergeeva

King

2013

Cell Stress and Chaperones

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“…Although direct structural information on the thermosome:substrate interaction is lacking, other structural and biochemical studies indicate that the working mechanism of the thermosomes follows the same lines as that of Group I (GroEL), that is, the release of trapped substrate after closure of the chaperonin cavity [73,77].…”

Section: The Folding Mechanismmentioning

confidence: 99%

“…This finding reinforces the concept that there are no steric impediments to simultaneous closure of both rings, a structural feature that could help to explain the kinetic behavior differences between the two chaperonin groups. An interesting characteristic of the Group II chaperonins is the role of the helical protrusion in the control of inter-ring communication [87,88]; the helices interact and stabilize the closed conformation [77,87]. Whereas the inter-ring region in Group I chaperonins shows considerable rigidity during the movement between closed and open states, that of the thermosomes undergoes large conformational changes, with the equatorial domains experiencing a $35°tilt and breaking of several salt bridges that stabilize the closed conformation [89].…”

Section: Conformational Changes and Reaction Cyclementioning

confidence: 99%

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Dynamics, flexibility, and allostery in molecular chaperonins

et al. 2015

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a b s t r a c tThe chaperonins are a family of molecular chaperones present in all three kingdoms of life. They are classified into Group I and Group II. Group I consists of the bacterial variants (GroEL) and the eukaryotic ones from mitochondria and chloroplasts (Hsp60), while Group II consists of the archaeal (thermosomes) and eukaryotic cytosolic variants (CCT or TRiC). Both groups assemble into a dual ring structure, with each ring providing a protective folding chamber for nascent and denatured proteins. Their functional cycle is powered by ATP binding and hydrolysis, which drives a series of structural rearrangements that enable encapsulation and subsequent release of the substrate protein.Chaperonins have elaborate allosteric mechanisms to regulate their functional cycle. Long-range negative cooperativity between the two rings ensures alternation of the folding chambers. Positive intra-ring cooperativity, which facilitates concerted conformational transitions within the protein subunits of one ring, has only been demonstrated for Group I chaperonins. In this review, we describe our present understanding of the underlying mechanisms and the structurefunction relationships in these complex protein systems with a particular focus on the structural dynamics, allostery, and associated conformational rearrangements.

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“…Mm-cpn molecules undergo large conformational changes during their functional cycle and can exist in a mixture of conformations under low ATP concentration (4,(28)(29)(30)(31). Moreover, the open-state Mm-cpn exhibits a dominant end-on view in cryo-EM, making it an excellent benchmark for tackling the preferred orientation problem (4).…”

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confidence: 99%

Multiscale natural moves refine macromolecules using single-particle electron microscopy projection images

Zhang

Mináry

Levitt

2012

Proc. Natl. Acad. Sci. U.S.A.

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The method presented here refines molecular conformations directly against projections of single particles measured by electron microscopy. By optimizing the orientation of the projection at the same time as the conformation, the method is well-suited to twodimensional class averages from cryoelectron microscopy. Such direct use of two-dimensional images circumvents the need for a three-dimensional density map, which may be difficult to reconstruct from projections due to structural heterogeneity or preferred orientations of the sample on the grid. Our refinement protocol exploits Natural Move Monte Carlo to model a macromolecule as a small number of segments connected by flexible loops, on multiple scales. After tests on artificial data from lysozyme, we applied the method to the Methonococcus maripaludis chaperonin. We successfully refined its conformation from a closed-state initial model to an open-state final model using just one class-averaged projection. We also used Natural Moves to iteratively refine against heterogeneous projection images of Methonococcus maripaludis chaperonin in a mix of open and closed states. Our results suggest a general method for electron microscopy refinement specially suited to macromolecules with significant conformational flexibility. The algorithm is available in the program Methodologies for Optimization and Sampling In Computational Studies.2D projection | structure refinement | stochastic optimization R ecent advances in single-particle cryoelectron microscopy, or cryo-EM, have enabled 3D structure determination of macromolecules to near-atomic resolution without crystallization, provided that the sample particles are homogeneous and adopt the same conformation (1-6). However, macromolecules are generally flexible in solution and can adopt multiple conformations in order to carry out their functions. Therefore, their cryo-EM images usually represent a heterogeneous mixture of macromolecular conformations. As a result, the power of single particle cryo-EM is limited, in that the reconstruction of a highresolution 3D density map requires hundreds of thousands to millions of particle images of the same conformation.Supervised classification (7) and maximum-likelihood methods (8) have been used to identify multiple structures from samples in which macromolecules experience moderate conformational fluctuations or exist in a small number of discrete structural states. Other approaches based on statistical bootstrapping (9, 10) have been used to separate different substrate-binding modes of macromolecules or to define flexible fragments within a molecular complex. However, these single-particle image processing techniques are severely limited when there are large conformational changes or nondiscrete conformational states (11, 12) that prevent correct determination of the orientation parameters for each raw particle image. Various computational techniques have been developed to model large conformational changes by flexible-fitting of a molecular model into the density map of a...

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Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber

Cited by 98 publications

References 37 publications

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Dynamics, flexibility, and allostery in molecular chaperonins

Multiscale natural moves refine macromolecules using single-particle electron microscopy projection images

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