An ensemble of cryo-EM structures of TRiC reveal its conformational landscape and subunit specificity.

TRiC/CCT assists the folding of ∼10% of cytosolic proteins through an ATP-driven conformational cycle and is essential in maintaining protein homeostasis. Here, we determined an ensemble of cryo-electron microscopy (cryo-EM) structures of yeast TRiC at various nucleotide concentrations, with 4 open-state maps resolved at near-atomic resolutions, and a closed-state map at atomic resolution, revealing an extra layer of an unforeseen N-terminal allosteric network. We found that, during TRiC ring closure, the CCT7 subunit moves first, responding to nucleotide binding; CCT4 is the last to bind ATP, serving as an ATP sensor; and CCT8 remains ADP-bound and is hardly involved in the ATPase-cycle in our experimental conditions; overall, yeast TRiC consumes nucleotide in a 2-ring positively coordinated manner. Our results depict a thorough picture of the TRiC conformational landscape and its allosteric transitions from the open to closed states in more structural detail and offer insights into TRiC subunit specificity in ATP consumption and ring closure, and potentially in substrate processing.

Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography.

Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT). This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. The aggregation of mHTT is inhibited in vitro and in vivo by the TCP-1 ring complex (TRiC) chaperonin. Recently, a novel complex comprised of a single type of TRiC subunit has been reported to inhibit mHTT aggregation. Specifically, the purified CCT5 homo-oligomer complex, when compared with TRiC, has a similar structure, ATP use, and substrate refolding activity, and, importantly, it also inhibits mHTT aggregation. Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (∼ 1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines (mHTTQ46-Ex1). We find that, in a similar fashion to TRiC, synthetic CCT5 complex caps mHTT fibrils at their tips and encapsulates mHTT oligomers, providing a structural description of the inhibition of mHTTQ46-Ex1 by CCT5 complex and a shared mechanism of mHTT inhibition between TRiC chaperonin and the CCT5 complex: cap and contain.

Structures of the Gß-CCT and PhLP1-Gß-CCT complexes reveal a mechanism for G-protein ß-subunit folding and Gßγ dimer assembly.

G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein ßγ (Gßγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gß and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gßγ assembly process, the Gß-CCT and the PhLP1-Gß-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gß interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gß with the CCTγ subunit. PhLP1 binding stabilizes the Gß fold, disrupting interactions with CCT and releasing a PhLP1-Gß dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.

Human CCT4 and CCT5 chaperonin subunits expressed in Escherichia coli form biologically active homo-oligomers.

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.