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

Abstract

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.

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

“…S3 D and E ). Consistent with our results, Chaetomium thermophilum TRiC, but with ATPase-deficient CCT8, has been shown to exhibit an ATPase activity very similar to that of the WT TRiC (43). Also, CCT8 is distinct from other subunits in the ATP binding-related sites, which are well conserved (39).…”

Intraring allostery controls the function and assembly of a hetero‐oligomeric class II chaperonin

“…This is mainly due to the intrinsic structural complexity of class II chaperonins, whose hetero-oligomeric nature makes a recombinant approach particularly challenging. As a matter of fact, Yamamoto et al (10) only recently succeeded in producing sufficiently ABBREVIATIONS: BUDE, Bristol University Docking Engine; CCT, chaperonin containing TCP-1; Cryo-EM, cryo-electron microscopy; MD, molecular dynamics; RhaD, rhamnose dehydrogenase; Ta, Thermoplasma acidophilum; WT, wild type large amounts of CCT from a thermophilic fungus in Escherichia coli for recombinant analysis. Indeed, most of the information available to date comes from in vitro studies conducted on CCT purified from mammalian and yeast cells.…”

Expression, Functional Characterization, and Preliminary Crystallization of the Cochaperone Prefoldin from the Thermophilic Fungus Chaetomium thermophilum

“…Indeed, several studies on C. thermophilum proteins have been reported [ 20 , 21 ]. Recently, we have succeeded in expressing CCT from C. thermophilum (CtCCT) in E. coli [ 19 ]. As expected, CtCCT exhibited a higher structural stability compared with mammalian or yeast CCTs.…”

“…In contrast, eukaryotic PFD and group II chaperonin (CCT/TRiC) require very complex structures consisting of eight and six different subunits, respectively, which makes it difficult to perform detailed biochemical and biophysical studies. Recently, we have succeeded in expression, purification, and characterization of CCT from a thermophilic fungus, C. thermophilum (CtCCT) [ 19 ]. Its thermal stability enabled us to obtain unprecedented information on CCT.…”

“…Recently, we succeeded in obtaining recombinant CCT from a thermophilic fungus, Chaetomium thermophilum (CtCCT) [ 19 ]. We obtained the recombinant CtCCT with a relatively high yield, and it exhibited a fairly high thermal stability.…”