Inter-Ring Communication Is Dispensable in the Reaction Cycle of Group II Chaperonins

Yamamoto, Yohei; Abe, Yuki; Moriya, Kazuki; Arita, Mayuno; Noguchi, Keiichi; Ishii, Noriyuki; Sekiguchi, Hiroshi; Sasaki, Y.; Yohda, Masafumi

doi:10.1016/j.jmb.2014.05.013

Cited by 11 publications

(9 citation statements)

References 29 publications

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“…The various CtCCT variants were constructed for further research. Previously, we analyzed the ATP-dependent motion of TKS1-Cpn at the single molecule level by DXT [10, 21, 22]. Gold nanocrystals were used as tracers for the subunit motion of CtCCT.…”

Section: Resultsmentioning

confidence: 99%

“…A UV light-triggered diffracted X-ray tracking (DXT) study with caged-ATP and stopped-flow fluorometry revealed that the lid was partially closed within 1 s after ATP binding, and the closed ring subsequently twisted counterclockwise within 2–6 s, as viewed from the top to bottom of TKS1-Cpn, and the twisted ring reverted to the original open state with a clockwise motion [21]. In addition, the intra-ring and inter-ring communications were studied using various mutant complexes of TKS1-Cpn [22, 23]. Studies of TKS1-Cpn complexes containing mutant subunits have shown that helical protrusion plays an important role in intra-ring cooperativity.…”

Section: Introductionmentioning

confidence: 99%

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Asymmetry in the function and dynamics of the cytosolic group II chaperonin CCT/TRiC

et al. 2017

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The eukaryotic group II chaperonin, the chaperonin-containing t-complex polypeptide 1 (CCT), plays an important role in cytosolic proteostasis. It has been estimated that as much as 10% of cytosolic proteins interact with CCT during their folding process. CCT is composed of 8 different paralogous subunits. Due to its complicated structure, molecular and biochemical investigations of CCT have been difficult. In this study, we constructed an expression system for CCT from a thermophilic fungus, Chaetomium thermophilum (CtCCT), by using E. coli as a host. As expected, we obtained recombinant CtCCT with a relatively high yield, and it exhibited fairly high thermal stability. We showed the advantages of the overproduction system by characterizing CtCCT variants containing ATPase-deficient subunits. For diffracted X-ray tracking experiment, we removed all surface exposed cysteine residues, and added cysteine residues at the tip of helical protrusions of selected two subunits. Gold nanocrystals were attached onto CtCCTs via gold-thiol bonds and applied for the analysis by diffracted X-ray tracking. Irrespective of the locations of cysteines, it was shown that ATP binding induces tilting motion followed by rotational motion in the CtCCT molecule, like the archaeal group II chaperonins. When gold nanocrystals were attached onto two subunits in the high ATPase activity hemisphere, the CtCCT complex exhibited a fairly rapid response to the motion. In contrast, the response of CtCCT, which had gold nanocrystals attached to the low-activity hemisphere, was slow. These results clearly support the possibility that ATP-dependent conformational change starts with the high-affinity hemisphere and progresses to the low-affinity hemisphere.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Asymmetry in the function and dynamics of the cytosolic group II chaperonin CCT/TRiC

et al. 2017

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“…The diffraction spots from each nanocrystal were monitored using an X-ray image intensifier (150 mm in diameter, V5445P, Hamamatsu photonics, Japan) and a CMOS camera (1024 pixel × 1024 pixel, SA 1.1, Photoron, Japan) at room temperature (25°C), and temperature was not controlled during the experiment. There is no critical temperature increase due to the incident X-rays because the functional motion of the protein, bacteriorhodopsin and cheperonin could be measured without cooling devices in previous DXT experiment73334. The specimen-to-camera distance was approximately 100 mm, and it was calibrated by diffraction from the gold film.…”

Section: Methodsmentioning

confidence: 99%

Real Time Ligand-Induced Motion Mappings of AChBP and nAChR Using X-ray Single Molecule Tracking

Sekiguchi

Suzuki

Nishino

et al. 2014

Sci Rep

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We observed the dynamic three-dimensional (3D) single molecule behaviour of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) using a single molecule tracking technique, diffracted X-ray tracking (DXT) with atomic scale and 100 μs time resolution. We found that the combined tilting and twisting motions of the proteins were enhanced upon acetylcholine (ACh) binding. We present the internal motion maps of AChBP and nAChR in the presence of either ACh or α-bungarotoxin (αBtx), with views from two rotational axes. Our findings indicate that specific motion patterns represented as biaxial angular motion maps are associated with channel function in real time and on an atomic scale.

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“…They share similar quaternary structures consisting of a double toroid cylinder assembled into two rings stacked back to back. This arrangement generates two dynamic internal cavities that allow unfolded proteins to fold correctly in a confined environment that opens and shuts in a cycle powered by ATP hydrolysis 11 12 . Group I Cpns are composed of 14 identical subunits arranged in two heptameric rings and require a co-chaperone, GroES, to facilitate protein folding.…”

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A human CCT5 gene mutation causing distal neuropathy impairs hexadecamer assembly in an archaeal model

Min

Angileri

Luo

et al. 2014

Sci Rep

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Chaperonins mediate protein folding in a cavity formed by multisubunit rings. The human CCT has eight non-identical subunits and the His147Arg mutation in one subunit, CCT5, causes neuropathy. Knowledge is scarce on the impact of this and other mutations upon the chaperone's structure and functions. To make progress, experimental models must be developed. We used an archaeal mutant homolog and demonstrated that the His147Arg mutant has impaired oligomeric assembly, ATPase activity, and defective protein homeostasis functions. These results establish for the first time that a human chaperonin gene defect can be reproduced and studied at the molecular level with an archaeal homolog. The major advantage of the system, consisting of rings with eight identical subunits, is that it amplifies the effects of a mutation as compared with the human counterpart, in which just one subunit per ring is defective. Therefore, the slight deficit of a non-lethal mutation can be detected and characterized.

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Inter-Ring Communication Is Dispensable in the Reaction Cycle of Group II Chaperonins

Cited by 11 publications

References 29 publications

Asymmetry in the function and dynamics of the cytosolic group II chaperonin CCT/TRiC

Asymmetry in the function and dynamics of the cytosolic group II chaperonin CCT/TRiC

Real Time Ligand-Induced Motion Mappings of AChBP and nAChR Using X-ray Single Molecule Tracking

A human CCT5 gene mutation causing distal neuropathy impairs hexadecamer assembly in an archaeal model

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