Molecular determinants of the ATP hydrolysis asymmetry of the CCT chaperonin complex

Chagoyen, Mónica; Carrascosa, José L.; Pazos, Florencio; Valpuesta, José M.

doi:10.1002/prot.24510

Cited by 23 publications

(24 citation statements)

References 33 publications

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“…S2), thereby providing estimates for the values of E a /R from the slopes and indicating that there is no change in the reaction mechanism in the temperature range of the measurements. The in vivo effects of mutations (16) and the relative affinities for ATP (17) were used in previous studies (13,18) to classify the ATPase activities of the different subunits into weak and strong. The data we collected show that the mutations in subunits CCT6, CCT3, and CCT7 caused the largest increases in the activation energy, which are about 3, 2, and 2 kcal·mol −1 , respectively.…”

Section: Resultsmentioning

confidence: 99%

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis

Gruber

Levitt

Horovitz

2017

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

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Knowing the mechanism of allosteric switching is important for understanding how molecular machines work. The CCT/TRiC chaperonin nanomachine undergoes ATP-driven conformational changes that are crucial for its folding function. Here, we demonstrate that insight into its allosteric mechanism of ATP hydrolysis can be achieved by Arrhenius analysis. Our results show that ATP hydrolysis triggers sequential "conformational waves." They also suggest that these waves start from subunits CCT6 and CCT8 (or CCT3 and CCT6) and proceed clockwise and counterclockwise, respectively.chaperonins | allostery | conformational changes | molecular machines A TP-fueled ring-shaped nanomachines are ubiquitous and found in all domains of life. Examples for such machines include chaperones that mediate protein folding and degradation (1), DNA and RNA remodeling enzymes (2), and proteins involved in intracellular trafficking (3). The oligomeric rings that form these machines usually consist of five to eight subunits that undergo coordinated conformational changes driven by ATP binding and/or hydrolysis. These conformational changes can take place in a concerted fashion as in the case of the chaperonin GroEL (4). Alternatively, they can also occur in a sequential or stochastic manner as reported, for example, for the CCT/TRiC chaperone complex (4) and ClpX (5), respectively. Knowing the mode of allosteric switching is essential for understanding the mechanism of action of biomolecular machines. Distinguishing between these different modes of allosteric switching can be achieved using native mass spectrometry (6, 7) and single-molecule fluorescence (8) and force-based (2) techniques but has been difficult to accomplish using traditional biochemical approaches. Here, we show that classical Arrhenius analysis can be used to unpick the allosteric mechanism of ATP hydrolysis by the CCT/ TRiC chaperone.CCT/TRiC is a member of group II chaperonins found in archaea and the eukaryotic cytosol that assist protein folding in an ATPdependent manner. Clients of this chaperonin system include β-actin, α-and β-tubulin, and several hundred other proteins (9, 10). CCT/TRiC consists of two identical back-to-back stacked octameric rings with a cavity at each end where protein folding can take place (11). Each ring of CCT/TRiC is made up of eight different subunits that are arranged in a fixed order around the ring. The correct order was established by determining which arrangement is most consistent with interresidue distance constraints obtained from chemical crosslinking and mass spectrometry (12). This order was also found to give the best fit to the crystallographic data for CCT/TRiC (9) and was, therefore, used to redetermine its structure accordingly (13). The eight subunits of CCT/TRiC have a similar structure that consists of three domains: (i) an apical domain that is involved in protein substrate binding, (ii) an equatorial domain that is involved in ring-ring interactions and contains an ATP binding site, and (iii) an intermediate domain that l...

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Section: Resultsmentioning

confidence: 99%

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis

Gruber

Levitt

Horovitz

2017

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

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“…Our approach focused on T-complex 1 (TCP-1) ring complex (TRiC), a cytosolic chaperonin (16,17) that participates in folding ∼10% of the cellular proteome (18,19). It also supports degradation of misfolded proteins through the proteasome and the autophagosome/lysosome pathways.…”

mentioning

confidence: 99%

TRiC subunits enhance BDNF axonal transport and rescue striatal atrophy in Huntington’s disease

Zhao

Chen

Han

et al. 2016

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

101

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Corticostriatal atrophy is a cardinal manifestation of Huntington's disease (HD). However, the mechanism(s) by which mutant huntingtin (mHTT) protein contributes to the degeneration of the corticostriatal circuit is not well understood. We recreated the corticostriatal circuit in microfluidic chambers, pairing cortical and striatal neurons from the BACHD model of HD and its WT control. There were reduced synaptic connectivity and atrophy of striatal neurons in cultures in which BACHD cortical and striatal neurons were paired. However, these changes were prevented if WT cortical neurons were paired with BACHD striatal neurons; synthesis and release of brain-derived neurotrophic factor (BDNF) from WT cortical axons were responsible. Consistent with these findings, there was a marked reduction in anterograde transport of BDNF in BACHD cortical neurons. Subunits of the cytosolic chaperonin T-complex 1 (TCP-1) ring complex (TRiC or CCT for chaperonin containing TCP-1) have been shown to reduce mHTT levels. Both CCT3 and the apical domain of CCT1 (ApiCCT1) decreased the level of mHTT in BACHD cortical neurons. In cortical axons, they normalized anterograde BDNF transport, restored retrograde BDNF transport, and normalized lysosomal transport. Importantly, treating BACHD cortical neurons with ApiCCT1 prevented BACHD striatal neuronal atrophy by enhancing release of BDNF that subsequently acts through tyrosine receptor kinase B (TrkB) receptor on striatal neurons. Our findings are evidence that TRiC reagentmediated reductions in mHTT enhanced BDNF delivery to restore the trophic status of BACHD striatal neurons., a genetically defined progressive neurodegenerative disorder, is characterized by abnormal involuntary movements, cognitive disability, and psychiatric symptoms (1). The pathogenesis of HD is due to an expanded CAG repeat in the huntingtin (HTT) gene, which encodes the protein Htt. Mutant Htt (mHTT) features an increased number of glutamines (Q > 36) near the N terminus. The striking atrophy and loss of striatal medium-sized spiny neurons (MSNs) in the HD brain are accompanied by atrophy of the cortex (2). The mechanisms by which mHTT induces dysfunction and death of neurons are actively explored (3). It has been shown that mHTT has an impact on axonal trafficking and signaling, synaptic function, gene expression, mitochondrial function, calcium homeostasis, and proteostasis (4, 5). Thus, mHTT plays a central role in HD pathogenesis, an assertion fully supported by recent studies in the BACHD model of HD (6).One productive line of inquiry regarding HD pathogenesis focuses on brain-derived neurotrophic factor (BDNF), which sustains neurons of the corticostriatal circuit (7-10). In the corticostriatal circuit, BDNF is produced in cortical neurons and is transported anterogradely in axons before secretion in striatum. Released BDNF binds to tyrosine receptor kinase B (TrkB) on striatal MSNs, resulting in BDNF-mediated signaling. Because MSNs do not produce BDNF, they are largely dependent on cortical neurons for i...

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“…A schematic representation of the asymmetry of CCT subunits is shown in layers (from outer to inner; adapted from Ref. [101]). Outer circle (black): possible CCT evolution pathway starting from CCT6 and proceeding through adjacent subunits to CCT2 [101].…”

Section: Evolution Of Group II Chaperoninsmentioning

confidence: 99%

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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Molecular determinants of the ATP hydrolysis asymmetry of the CCT chaperonin complex

Cited by 23 publications

References 33 publications

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis

TRiC subunits enhance BDNF axonal transport and rescue striatal atrophy in Huntington’s disease

Dynamics, flexibility, and allostery in molecular chaperonins

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