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4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement - PubMed

  • ️Fri Jan 01 2010

. 2010 Mar 16;107(11):4967-72.

doi: 10.1073/pnas.0913774107. Epub 2010 Mar 1.

Affiliations

4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Yao Cong et al. Proc Natl Acad Sci U S A. 2010.

Abstract

The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of approximately 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-A resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-A resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Calpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed approximately 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

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Conflict of interest statement

Conflict of interest statement: The Sponsor is an investigator of the Nanomedicince Development Center with the support of National Institutes of Health Grant (5PN2EY016525), which is directed by the corresponding author.

Figures

Fig. 1.
Fig. 1.

TRiC 4.7 Å asymmetric cryo-EM density map and the identification of the 2-fold axis between its two rings. (A) A typical 300 kV image of ice embedded TRiC in the both-ring-closed conformation. Representative particles are highlighted by white boxes. (B) End-on view of a ring and side view of both rings of TRiC with different subunits in different colors. The identified 2-fold axis is indicated by black ellipsoid in the side view. (C) Rotational correlation coefficient analysis between a ring and b ring. At 0° the two rings have the best correlation score, indicating the location of the 2-fold axis as illustrated in B. Due to the periodicity of the TRiC map, 0° peak of the curve is split, with the other half of the peak at ∼359°. (D) Cartoon diagram depicts the relative orientation of the two rings of TRiC.

Fig. 2.
Fig. 2.

The match of the side-chain densities in an apical domain region with the corresponding optimized model for each of the eight subunits. (A) Location of this stretch (Sky Blue, including the protruding helix H8 and the connected loop) in a complete TRiC subunit (Gray). The three domains are labeled. (B) Sequence alignment of bovine TRiC eight subunits in that apical domain region as shown in A. Unique sequence stretches of each subunit are highlighted by red characters. Either one or a combination of several such characteristic stretches can serve as a fingerprint for each TRiC subunit. (C) Subunit ai map (Blue meshes) with the optimized model of the best matching CCT8(θ) (Red). The residues with the clearly observable side-chain densities are labeled in black or red. Here the red labels correspond to the residues in the unique stretches of CCT8(θ) as in B. (D–J) Similar rendering style as in C for each of the subunit maps and the corresponding optimized models in the equivalent region.

Fig. 3.
Fig. 3.

The optimized Cα model of the entire TRiC complex with its 16 subunits in the spatial arrangement determined by our cryo-EM structure. (A) End-on and side views of the TRiC complex. Same color scheme as in Fig. 1. (B) Cartoon diagram illustrating the arrangement of the 16 subunits in the two rings. We denote the TRiC subunits only by their CCT number. (C) Summary of near neighbors TRiC subunits identified by chemical cross-linking. Neighboring subunit pairs consistent with the indicated models are marked with a “+,” and those inconsistent with the indicated models are marked with a “−.”

Fig. 4.
Fig. 4.

The atomic model of CCT2(β) subunit and the match between the aiii density and the model. (A) Optimized atomic model of CCT2(β) with the N terminus in blue and C terminus in red. Zoom in views show the match between three stretches of densities highlighted in different color frames and the corresponding model. (B) The Ramachandran plot of the CCT2(β) model calculated by MolProbity (48) shows that over 95% of the dihedral angles fall within allowable regions.

Fig. 5.
Fig. 5.

Surface property of the central cavity of TRiC, thermosome (1A6E) and the cis-ring of GroEL-GroES (1AON). (A–C) The inner cavity electrostatic potential of the TRiC/thermosome/GroEL-GroES are shown in cutaway views of the apical and intermediate domains, including GroES for C. Blue represents positively charged patches, red negatively charged patches, and white neutral patches. The smaller panel illustrates the viewing angle. Of note, the surfaces are approximate and variances due to model/map resolution may affect the fine details of surface potential. (D–F) Inner wall surface property of TRiC/thermosome/GroEL-GroES with side-chain properties is shown: hydrophilic (Sky Blue), hydrophobic (Yellow), and main chain (White).

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