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.
Highlights
Chaperones are proteins that bind to unfolded or misfolded polypeptides and induce correct folding or facilitate degradation [1, 2]
The variation among the containing t-complex polypeptide 1 (CCT) subunits should be important for the function of CCT
We examined the dynamics of CtCCT by diffracted X-ray tracking (DXT) and revealed an asymmetry in ATP-induced motion in a ring
Summary
Chaperones are proteins that bind to unfolded or misfolded polypeptides and induce correct folding or facilitate degradation [1, 2]. The protein-folding mechanism mediated by group II chaperonins has been studied by using simpler archaeal homologues [11, 13,14,15,16]. The mechanism of ATP-dependent conformational change for group II chaperonins from an open to closed conformation has been studied using archaeal chaperonins. The asymmetric ring complex exhibited ATP-dependent conformational change and protein folding activity as well, which demonstrated that inter-ring communication was dispensable in the reaction cycle of group II chaperonins. The eukaryotic group II chaperonin, the chaperonin-containing t-complex polypeptide 1 (CCT, known as TRiC, TCP-1 ring Complex), is different from archaeal homologues. Archaeal group II chaperonins function as general chaperones by capturing denatured protein through hydrophobic interactions, CCT appears to have substrate specificity. We examined the dynamics of CtCCT by DXT and revealed an asymmetry in ATP-induced motion in a ring
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