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Staggered ATP binding mechanism of eukaryotic chaperonin TRiC (CCT) revealed through high-resolution cryo-EM

Nature Structural & Molecular Biology volume 23, pages 1083–1091 (2016)Cite this article

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Abstract

The eukaryotic chaperonin TRiC (or CCT) assists in the folding of 10% of cytosolic proteins. Here we present two cryo-EM structures of Saccharomyces cerevisiae TRiC in a newly identified nucleotide partially preloaded (NPP) state and in the ATP-bound state, at 4.7-Å and 4.6-Å resolution, respectively. Through inner-subunit eGFP tagging, we identified the subunit locations in open-state TRiC and found that the CCT2 subunit pair forms an unexpected Z shape. ATP binding induces a dramatic conformational change on the CCT2 side, thereby suggesting that CCT2 plays an essential role in TRiC allosteric cooperativity. Our structural and biochemical data reveal a staggered ATP binding mechanism of TRiC with preloaded nucleotide on the CCT6 side of NPP-TRiC and demonstrate that TRiC has evolved into a complex that is structurally divided into two sides. This work offers insight into how the TRiC nucleotide cycle coordinates with its mechanical cycle in preparing folding intermediates for further productive folding.

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Figure 1: Cryo-EM maps of TRiC in the NPP and ATP-bound states.
Figure 2: Determination of subunit locations in NPP-TRiC and the match between the pseudoatomic model and the corresponding map.
Figure 3: Conformational change in TRiC triggered by ATP binding.
Figure 4: Nucleotide distribution analysis of different states of TRiC.
Figure 5: Changes in inter-ring and intraring interaction patterns induced by ATP binding.
Figure 6: Proposed staggered ATP binding and substrate-folding mechanisms for TRiC.

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Acknowledgements

We thank W. Chiu (Baylor College of Medicine) for critical discussion of this work, and J. Zhou and Z. Zhou (Institute of Biochemistry and Cell Biology) for their generous support. We thank J. Li, Y. Zhang, and M. Cao from the EM facility and managers from the Database and Computing facility of NCPSS for their assistance with the EM instruments and parallel computing. We are grateful to the NCPSS Protein Expression and Purification Facility, Mass Spectrometry Facility, Nuclear Magnetic Resonance Facility, and Integrated Laser Microscopy Facility for instrument support and technical assistance. This work was supported by grants to Y.C. from the CAS Pilot Strategic Science and Technology Projects B (XDB08030201), the National Basic Research Program of China (2013CB910401), the National Natural Science Foundation of China (31270771 and 31222016), the STS program of the CAS (KFJ-EW-STS-098), and the CAS–Shanghai Science Research Center (CAS-SSRC-YH-2015-01).

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  1. Yunxiang Zang, Mingliang Jin and Huping Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. National Center for Protein Science Shanghai, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Yunxiang Zang, Mingliang Jin, Huping Wang, Zhicheng Cui, Liangliang Kong, Caixuan Liu & Yao Cong

  2. University of Chinese Academy of Sciences, Beijing, China

    Yunxiang Zang, Mingliang Jin, Huping Wang & Caixuan Liu

  3. Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai, China

    Liangliang Kong & Yao Cong

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Contributions

Y.C. and Y.Z. designed the experiments; Y.Z. purified the proteins and optimized the cryo-EM sample-preparation conditions, and M.J., H.W., Z.C., and C.L. were also involved in the protein purification; Y.Z., M.J., and H.W. collected the cryo-EM data with assistance from L.K.; Y.Z. and M.J. performed the cryo-EM reconstruction; Y.Z., M.J., and Y.C. analyzed the data and wrote the paper.

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Correspondence to Yao Cong.

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Integrated supplementary information

Supplementary Figure 1 Purification and characterization of the TRiC complex from S. cerevisiae.

(a) SDS-PAGE of the purified TRiC sample stained with Coomassie Brilliant Blue. The red rectangle highlights the bands belonging to the TRiC subunits. (b) The purified yeast TRiC remained active in a luciferase refolding assay. Yeast TRiC refolded an unfolded luciferase more efficiently than did BSA or buffer controls. (c) Representative cryo-EM images of NPP-TRiC and TRiC-AMP-PNP, respectively. Scales are labeled. (d) Reference-free 2D class averages of NPP-TRiC and TRiC-AMP-PNP, respectively.

Supplementary Figure 2 Resolution evaluation of the NPP-TRiC and TRiC–AMP-PNP cryo-EM maps.

(a) Resolution estimation of the NPP-TRiC (blue curve) and TRiC-AMP-PNP (red curve) maps according to the gold-standard FSC criterion of 0.143. (b) Local resolution estimation of the NPP-TRiC map by Resmap. The resolution color bar (in Å) is also labeled. (c) Local resolution estimation of the TRiC-AMP-PNP map.

Supplementary Figure 3 Comparison of the yeast and bovine TRiC conformations and visualization of the extra densities in the E-domain region of yeast TRiC.

(a) Alignment of the yeast NPP-TRiC (gold) and bovine1 apo-TRiC (gray) maps revealed distinct conformations in several subunits. (b) Alignment of the yeast (dodger blue) and bovine (gray) TRiC-AMP-PNP maps indicated similar conformations. The two yeast TRiC maps were low pass filtered to 5.5 Å resolution. (c,d) Zoom in view of the extra densities in the E domain region of the NPP-TRiC (c) and TRiC-AMP-PNP (d) maps in both the end-on view (left panel) and the side view (right panel). The two maps were both low pass filtered to 10 Å resolution to better visualize the two pieces of extra densities (in blue) in the E domain region, forming a blockage between the two chambers of TRiC. The maps are colored according to its cylinder radius following the same color scheme as in Fig. 1a,b. The connecting region between the extra density and the inner surface of TRiC is indicated by red dotted circle. In both maps, we can observe that each of the extra densities could potentially connect to the N-terminal region of the CCT3, CCT5′, and CCT7′ subunits.

1. Cong, Y. et al. Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle. EMBO J 31, 720-30 (2012).

Supplementary Figure 4 Analysis of the relative orientations of subunits CCT1, CCT4, and CCT6 between the two rings of TRiC, and CCT6 subunit identification in the CCT6-eGFP TRiC map in the open state.

The top row three 3D maps are of ATP-AlFx TRiC in the both-ring closed conformation. eGFP was used to tag (a) the E domain of CCT1, (b) I domain of CCT4, or (c) I domain of CCT6. The TRiC density is in gray, and the extra density protruding from the complex corresponding to the eGFP tag is colored in green throughout this figure. Three subunit pairs were found to separate the CCT1 subunits from each ring, whereas only one subunit pair was determined to separate the CCT4 subunits from each ring. The CCT6 subunits of the cis-ring and trans-ring directly contacted each other. These geometrical relationships between the two eGFP tags (one from each ring) are in line with that proposed in the XL-MS studies. (d) The end-on view and two side views of the CCT6-eGFP TRiC map in the open state. Two extra pieces of exposed densities were observed to be located in the I domain of the on-axis subunit a5 and a5′ positions, indicating a5 to be CCT6.

Supplementary Figure 5 Schematic drawing of the secondary-structure elements of a TRiC subunit.

Supplementary Figure 6 Identification of the residual nucleotide forms in the NPP-TRiC and TRiC–AMP-PNP samples on the basis of the luciferin-luciferase reaction with the ADP/ATP ratio assay.

(a) Nucleotide form identification of the NPP-TRiC sample. The relative light unit (RLU) values were measured for NPP-TRiC sample with or without proteinase K digestion, with NPP-TRiC sample buffer as negative control (NC) and 1 μM ATP and 1 μM ADP as positive control. This set of experiments demonstrated the presence of both ATP and ADP in the NPP-TRiC sample, with the ADP form being the much dominant population. Moreover, the proteinase K digested sample showed much higher RLU value compared to the undigested sample, indicating proteinase K digestion did help to release the residual nucleotide from the binding pockets. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments). (b) Nucleotide form identification of the TRiC-AMP-PNP sample. TRiC sample was incubated with 10 mM AMP-PNP, then the free AMP-PNP as well as the potentially replaced nucleotide from the TRiC sample were removed. We tested the RLU values for the AMP-PNP incubated sample with or without proteinase K digestion, as well as the AMP-PNP un-incubated sample with or without proteinase K digestion, with the AMP-PNP incubation buffer as negative control (NC). This experiment suggested the presence of both ATP and ADP in the TRiC-AMP-PNP sample, with the ADP form being the much dominant population. Additionally, the AMP-PNP incubated sample showed lower RLU value compared to the AMP-PNP un-incubated sample, indicating that part of the residual nucleotide can be replaced by the excess AMP-PNP. Moreover, there was detectable ADP in the filtrate, which could come from the broken TRiC particles caused by the repeated dilution and concentration in the buffer exchange process in all the experiments in b. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments). (c) As a control experiment, the RLU values of ATP and ADP at different concentrations were also measured using the same assay. Error bars indicate SD; n=3 technical replicates of a representative experiment (out of three experiments).

Supplementary Figure 7 Conformation comparison between the 3D reconstructions with a fully asymmetric initial model or a C8-symmetry-imposed initial model.

The resulting 3D map (light green) from the completely asymmetric initial model showed a conformation almost identical to that of our 4.7 Å resolution map (gold) using the C8 symmetrized map as the initial model.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1231 kb)

Conformational transition of TRiC triggered by ATP binding.

Conformational changes of TRiC complex triggered by ATP-binding are show in the top and four different side views. (MOV 13284 kb)

Conformational change of CCT2, CCT5, and CCT7 triggered by ATP binding.

The conformational change of TRiC subunit CCT2, CCT5, CCT7 triggered by ATP-binding is shown in the side view, respectively. (MOV 13638 kb)

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Zang, Y., Jin, M., Wang, H. et al. Staggered ATP binding mechanism of eukaryotic chaperonin TRiC (CCT) revealed through high-resolution cryo-EM. Nat Struct Mol Biol 23, 1083–1091 (2016). https://doi.org/10.1038/nsmb.3309

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