Kazuki Moriya

ATP Dependent Rotational Motion of Group II Chaperonin Observed by X-ray Single Molecule Tracking

Group II chaperonins play important roles in protein homeostasis in the eukaryotic cytosol and in Archaea. These proteins assist in the folding of nascent polypeptides and also refold unfolded proteins in an ATP-dependent manner. Chaperonin-mediated protein folding is dependent on the closure and opening of a built-in lid, which is controlled by the ATP hydrolysis cycle. Recent structural studies suggest that the ring structure of the chaperonin twists to seal off the central cavity. In this study, we demonstrate ATP-dependent dynamics of a group II chaperonin at the single-molecule level with highly accurate rotational axes views by diffracted X-ray tracking (DXT). A UV light-triggered DXT study with caged-ATP and stopped-flow fluorometry revealed that the lid partially closed within 1 s of ATP binding, the closed ring subsequently twisted counterclockwise within 2–6 s, as viewed from the top to bottom of the chaperonin, and the twisted ring reverted to the original open-state with a clockwise motion. Our analyses clearly demonstrate that the biphasic lid-closure process occurs with unsynchronized closure and a synchronized counterclockwise twisting motion.

Dissection of the ATP-Dependent Conformational Change Cycle of a Group II Chaperonin☆

Group II chaperonin captures an unfolded protein while in its open conformation and then mediates the folding of the protein during ATP-driven conformational change cycle. In this study, we performed kinetic analyses of the group II chaperonin from a hyperthermophilic archaeon, Thermococcus sp. KS-1 (TKS1-Cpn), by stopped-flow fluorometry and stopped-flow small-angle X-ray scattering to reveal the reaction cycle. Two TKS1-Cpn variants containing a Trp residue at position 265 or position 56 exhibit nearly the same fluorescence kinetics induced by rapid mixing with ATP. Fluorescence started to increase immediately after the start of mixing and reached a maximum at 1-2s after mixing. Only in the presence of K(+) that a gradual decrease in fluorescence was observed after the initial peak. Similar results were obtained by stopped-flow small-angle X-ray scattering. A rapid fluorescence increase, which reflects nucleotide binding, was observed for the mutant containing a Trp residue near the ATP binding site (K485W), irrespective of the presence or absence of K(+). Without K(+), a small, rapid fluorescence decrease followed the initial increase, and then a gradual decrease was observed. In contrast, with K(+), a large, rapid fluorescence decrease occurred just after the initial increase, and then the fluorescence gradually increased. Finally, we observed ATP binding signal and also subtle conformational change in an ATPase-deficient mutant with K485W mutation. Based on these results, we propose a reaction cycle model for group II chaperonins.

Inter-ring communication is dispensable in the reaction cycle of group II chaperonins.

Chaperonins are ubiquitous molecular chaperones with the subunit molecular mass of 60kDa. They exist as double-ring oligomers with central cavities. An ATP-dependent conformational change of the cavity induces the folding of an unfolded protein that is captured in the cavity. In the group I chaperonins, which are present in eubacteria and eukaryotic organelles, inter-ring communication takes important role for the reaction cycle. However, there has been limited study on the inter-ring communication in the group II chaperonins that exist in archaea and the eukaryotic cytosol. In this study, we have constructed the asymmetric ring complex of a group II chaperonin using circular permutated covalent mutants. Although one ring of the asymmetric ring complex lacks ATPase or ATP binding activity, the other wild-type ring undergoes an ATP-dependent conformational change and maintains protein-folding activity. The results clearly demonstrate that inter-ring communication is dispensable in the reaction cycle of group II chaperonins.

Direct Observation of Torsional Motion of Group II Chaperonin

Diffracted X-ray Tracking (DXT) has been considered as a powerful technique in biological science for detecting subtle (pico meter scale) dynamic motion of the target protein at single molecular level. This method was applied for various proteins, such as bacteriorhodopsin [1], antibody [2] and KcsA channel [3]. In DXT, the dynamics of a single protein can be monitored through trajectory of the Laue spot from the nanocrystal which was labeled on the objective protein immobilized on the substrate surface.In this study, DXT method was applied to the group II chaperonin, a protein machinery that captures an unfolded protein and refolds it to the correct conformation in an ATP dependent manner [4]. A mutant group II chaperonin from Thermococcus strain KS-1 with a Cys residue at the tip of the helical protrusion, was immobilized on the gold coated substrate surface and was labeled with a gold nanocrystal through gold-thiol bond.We monitored diffracted spots from the nanocrystal as dynamic motion of the chaperonin, and found that the rotational motion of the nanocrystal, which corresponded to the torsional motion of the chaperonin, in the presence of ATP condition was 10 times larger than that in the absence of ATP condition. And UV-light triggered DXT study using caged ATP revealed that the chaperonin twisted counterclockwisely (from the top to the bottom view of chaperonin) when ATP binded to the chaperonin and the angular velocity from open to closed state of chaperonin chamber was 10 % faster than that from closed to open state.[1] Y. Okumura et al., Phys. Rev. E, 70:021917 (2004)[2] T. Sagawa et al., Biochem. Biophys. Res. Commun. 335:770 (2007)[3] H. Shimizu et al., Cell 132:67 (2008)[4] T. Kanzaki et al., J. Biol. Chem. 283: 34773 (2008)