Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT

Abstract

All chaperonins mediate ATP-dependent polypeptide folding by confining substrates within a central chamber. Intriguingly, the eukaryotic chaperonin TRiC (also called CCT) uses a built-in lid to close the chamber, whereas prokaryotic chaperonins use a detachable lid. Here we determine the mechanism of lid closure in TRiC using single-particle cryo-EM and comparative protein modeling. Comparison of TRiC in its open, nucleotide-free, and closed, nucleotide-induced states reveals that the interdomain motions leading to lid closure in TRiC are radically different from those of prokaryotic chaperonins, despite their overall structural similarity. We propose that domain movements in TRiC are coordinated through unique interdomain contacts within each subunit and, further, these contacts are absent in prokaryotic chaperonins. Our findings show how different mechanical switches can evolve from a common structural framework through modification of allosteric networks.

Figure 1

Cryo-EM density maps of the eukaryotic chaperonin TRiC in its open and closed conformations. (a) A representative area of a CCD-captured image of ice-embedded TRiC in the closed state, generated by incubation with ATP and AlF_x. (b,c) Side and top views of a single-particle reconstruction of TRiC in its closed state, carried out with 7,129 particles to ∼15-Å resolution. (d) A representative area of a CCD-captured image of ice-embedded open-state, nucleotide-free TRiC. (e,f) Side and top views of a single-particle reconstruction of TRiC in its open state, carried out with 13,287 particles to ∼18-Å resolution.

Figure 2

Comparative protein structure modeling the closed state of TRiC. (a) Homology model of the TRiC α subunit in the closed state. The apical domain is red, the intermediate domain is yellow and the equatorial domain is blue. (b) Fit of the TRiC α subunit model into the cryo-EM density map corresponding to a monomer of the closed state. (c,d) Top and side views of the homology model of closed TRiC docked into the cryo-EM density map. The fit has a similarity score of 0.987.

Figure 3

Comparative protein structure modeling the open state of TRiC. (a) Homology model of the TRiC α subunit in the open state. The apical domain is red, the intermediate domain is yellow and the equatorial domain is blue. The short apical segment that forms the lid in the closed state is highlighted in cyan. (b) The fit of the TRiC α subunit into the density map corresponding to a monomer of the open conformation. The short apical lid segment is absent in the open-state map, probably owing to its dynamic nature. (c,d) Top and side views of the open TRiC model docked into the open-state cryo-EM density map. The fit has a similarity score of 0.969. (e) Top view of the ATP-driven open-to-closed transition in TRiC.

Figure 4

Comparison of the conformational changes of TRiC and GroEL subunits. (a,b) Low-resolution representations of the TRiC subunits in the open, nucleotide-free (a) and closed, ATP-induced (b) conformations. The apical, intermediate and equatorial domains are shown in red, yellow and blue, respectively. Arrows indicate the direction and magnitude of proposed domain motions during lid closure. The intermediate domains rotate about ∼25° toward the equatorial domain; the apical domains undergo a rotation of ∼50° toward the equatorial domain accompanied by a lateral rotation of ∼50° into the central chamber. Stars mark the fixed positions in each domain to facilitate visualization of relative domain movements. (c,d) Low-resolution representations of a GroEL subunit in the open (nucleotide-free) (c) and closed (nucleotide-bound and GroES-bound) (d) conformations, colored as in a,b, with stars marking fixed positions in each domain, as above. Arrows show the domain motions upon nucleotide and GroES binding. The intermediate domains rotate about ∼25° toward the equatorial domain; the apical domains undergo a rotation of ∼60° away from the equatorial domain accompanied by a lateral rotation of ∼90° away from the central chamber.

Figure 5

Apical-intermediate domain interfaces from crystallographic structures of the thermosome and GroEL. (a) The α subunit of the TRiC-like chaperonin called the thermosome. The apical domain is red, the intermediate domain is yellow and the equatorial domain is blue. Inset, (viewed reversed relative to the subunit figures on the left) the contacts between the apical and intermediate domains are highlighted. (b,c) GroEL subunit in its closed, GroES-bound (b) and open, unliganded (c) conformations, with domains colored as for the thermosome. Insets (viewed reversed relative to the subunit figures on the left) indicate the lack of contacts between the apical and intermediate domains in either state.