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The cryo-EM structure of the chloroplast ClpP complex

Nature Plants volume 7, pages 1505–1515 (2021)Cite this article

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Abstract

Protein homoeostasis in plastids is strategically regulated by the protein quality control system involving multiple chaperones and proteases, among them the Clp protease. Here, we determined the structure of the chloroplast ClpP complex from Chlamydomonas reinhardtii by cryo-electron microscopy. ClpP contains two heptameric catalytic rings without any symmetry. The top ring contains one ClpR6, three ClpP4 and three ClpP5 subunits while the bottom ring is composed of three ClpP1C subunits and one each of the ClpR1–4 subunits. ClpR3, ClpR4 and ClpT4 subunits connect the two rings and stabilize the complex. The chloroplast Cpn11/20/23 co-chaperonin, a co-factor of Cpn60, forms a cap on the top of ClpP by protruding mobile loops into hydrophobic clefts at the surface of the top ring. The co-chaperonin repressed ClpP proteolytic activity in vitro. By regulating Cpn60 chaperone and ClpP protease activity, the co-chaperonin may play a role in coordinating protein folding and degradation in the chloroplast.

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Fig. 1: Co-chaperonins interact with CrClpP.
Fig. 2: Co-chaperonins inhibit ClpP proteolytic activity.
Fig. 3: Cryo-EM structure of the Chlamydomonas ClpP complex.
Fig. 4: Properties of the Chlamydomonas ClpP core complex.
Fig. 5: Cryo-EM structure of the Chlamydomonas ClpP-Cpn11/20/23 complex.

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Data availability

Electron density maps have been deposited in the Electron Microscopy Data Bank under accession codes EMD-31171 for CrClpP-S1, EMD-31175 for ClpP-S2, EMD-31173 for ClpP-S2c and EMD-31174 for Cpn11/20/23. Related atom coordinates file also has been submitted to the Protein Data Bank, with accession codes 7EKO for CrClpP-S1 and 7EKQ for CrClpP-S2c. Source data are provided with this paper.

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Acknowledgements

We are grateful to the staff of the NCPSS EM facility, Mass Spectrometry facility and Database and Computing facility for instrument support and technical assistance. This work was funded by the Strategic Priority Research Program of Chinese Academy of Sciences (grant nos. XDA24020103-2 and XDB37040103), the National Key Research and Development Program of China (2016YFD0100405 and 2017YFA0503503) and the Ministry of Agriculture of China (2016ZX08009-003-005), the ‘Initiative d’Excellence’ programme from the French State (grant ‘DYNAMO’, ANR-11-LABX-0011-01) and the DFG (TRR 175, project C02). We thank J. D. Rochaix and S. Ramundo for fruitful discussion.

Author information

Author notes

  1. These authors contributed equally: Ning Wang, Yifan Wang.

Authors and Affiliations

  1. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, China

    Ning Wang, Qian Zhao, Wenjuan Zhang, Yanan Liu & Cuimin Liu

  2. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

    Ning Wang, Yifan Wang, Wenjuan Zhang, Yanan Liu & Cuimin Liu

  3. State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China

    Yifan Wang, Xiang Zhang & Yao Cong

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

    Yifan Wang, Xiang Zhang & Yao Cong

  5. National Facility for Protein Science in Shanghai, Zhangjiang Lab, Shanghai Advanced Research Institute, CAS, Shanghai, China

    Chao Peng

  6. Institut de Biologie Physico-Chimique, Sorbonne Université, Paris, France

    Olivier Vallon

  7. Molecular Biotechnology & Systems Biology, TU Kaiserslautern, Kaiserslautern, Germany

    Michael Schroda

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Contributions

C.L. and Y.C. supervised the project. N.W. executed all biochemical experiments. Y.W. and X.Z. collected the cryo-EM data. Y.W. did data processing with initial map from X.Z. Y.W. and N.W. did model building and structural analysis. Q.Z. started the project and optimized the protein purification. C.P. performed the mass spectrometry analysis. W.Z. and Y.L. helped to purify protein. O.V. and M.S. were involved in the project design, data analysis and interpretation. C.L., N.W., O.V. and M.S. wrote the manuscript with modification from Y.W. and Y.C.

Corresponding authors

Correspondence to Yao Cong or Cuimin Liu.

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The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Oliver Martin Mueller-Cajar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Analysis of the CrClpP complex and its interaction with co-chaperonins.

Mass determination of purified CrClpP complexes by AFFFF. The horizontal blue line across the peak displays the molar mass.

Extended Data Fig. 2 Analysis of the proteolytic activities of ClpP complexes from E. coli and Chlamydomonas.

(a) Degradation of β-casein was monitored in reactions containing β-casein (16 μM), CrClpP (0.4 μM), EcClpP (0.4 μM), Cpn20 (0.4 μM), Cpn11/20/23 (0.4 μM) and ADEP dissolved in DMSO (4, 8 or 18 μM) as indicated. The reactions were performed at 30°C and aliquots taken at the indicated time points were analysed via SDS–PAGE (15% gels) and Coomassie staining. The position of β-casein and the EcClpP protein are indicated. The casein degradation products resulting from proteolytic attack by the CrClpP complex are shown in the dotted box. Individual degradation experiment was repeated at least three times independently with similar results and a representative result was shown. (b) Densitometric quantification of β-casein from the reactions with CrClpP and ClpP+GroES. Shown are mean values from three independent replicates, error bars represent SD. (c) Gel filtration of ClpP complexes in the presence or absence of ADEP. 1 μM ClpP, 2 μM co-chaperonin or 1 μM ClpP supplemented with 18 μM ADEP were injected into a Superdex 200 PC 3.2/10 column with 20 mM MOPS-KOH, pH 7.5, 80 mM NaCl, 10 mM MgCl2, 10 mM KCl, 1 mM DTT, 10% glycerol. The relevant factions were collected and analysed by western blot with Cpn20 antibodies. The experiment was repeated three times independently with similar results and a representative result was shown.

Source data

Extended Data Fig. 3 Data collection and processing of ClpP particles visualized by cryo-EM.

(a) Representative micrograph of CrClpP complexes from three independent experiments with similar results. The scale bar equals 100 nm. (b) 2D class averages of CrClpP complexes. (c) Workflow of the 3D reconstruction based on cryo-EM data. A total of 578,978 particles were used for 3D classification.

Extended Data Fig. 4 Statistics of the final density map of ClpP.

(a) Overview of ClpP-S2 particles. (b) Overview of Cpn11/20/23 particles. (c) Gold standard Fourier Shell Correlation (FSC) curves of the final refinement map of ClpP-S1, ClpP-S2 and Cpn11/20/23. (d) Local resolution map of ClpP-S1, ClpP-S2 and co-chaperonin densities. (e) The particle orientation distributions in the final iteration of structure refinement. Red parts in the cylinders represent more particles in this direction.

Extended Data Fig. 5 Alignment of the amino acid sequences of ClpP/R subunits.

Sequence alignment of ClpP and ClpR subunits from Escherichia coli (Ec), Synechocystis sp. PCC6803 (Sy), Mycobacterium tuberculosis (Mb), Chlamydomonas reinhardtii (Cr) and Arabidopsis thaliana (At), extracted form an alignment of 243 algal, plant and bacterial sequences. Structural elements derived from the crystal structure of E. coli ClpP are shown on top of the alignment (α: α-helices, β: β-strands). The three catalytic residues are marked by red arrows. The best-conserved residues are shown with a coloured background. Chlamydomonas subunits (names in red) are shown with the experimentally-determined mature N terminus boxed and sections not assigned in the Clp-S1 structure in faded colours. ClpP1_Cr is shown after removal of the insertion sequence IS1 (at pos 59, purple arrow), so the N terminus of ClpP1c, starting near the end of IS1, is not shown. The less conserved regions of Clp subunits used for subunit assignment in Fig. S6B are underlined with red lines. The proline motif region is underlined with a blue line.

Extended Data Fig. 6 Pseudo-atomic models of Clp subunits and close-up views of the fitting of long side chains of selected amino acids into the density map.

(a) Pseudo-atomic models of conserved sequences of Clp subunits. The sequences correspond roughly to amino acids 27 to 175 in EcClpP. Each colour ribbon represents a different Clp subunit. (b) Close-up views of the density maps accommodating long side chains of selected amino acids in individual ClpP/R subunits. The selected sequence regions are indicated in Fig. S5 and Fig. S6.

Extended Data Fig. 7 Model-to-map fitting.

(a) Density maps of Clp subunits. Ribbon presentations of the structural models of the individual subunits are docked into the cryo-EM densities with the subunit assignment indicated below. For each subunit, the N- and C-terminal residues of the assigned sequence are indicated. (b) Correlation coefficient (CC) value of each subunit in the ClpP core complex. Individual Clp subunit was labelled in X-axis. The CC value is generated by Phenix 1.19. (c) A superimposition of the subunit models (coloured) and the cryo-EM density map (grey). Unassigned, additional density maps A2 and A3 are encircled with broken black lines.

Extended Data Fig. 8 Properties of the ClpP-Cpn11/20/23 complex.

(a) A superimposition of the EcClpP complex with ClpP-S2 particles. The additional density map A4 is the co-chaperonin complex. (b) Correlation coefficient (CC) values of each subunit of the ClpP-Cpn11/20/23 complex. Individual Clp and co-chaperonin subunit was labelled in X-axis. The CC value is generated by Phenix 1.19. (c) Density maps of co-chaperonin subunits. Ribbon presentations of the structural models of the individual co-chaperonin subunits, which are docked into the cryo-EM densities. (d) Insertion of the mobile loops of Cpn20N into the hydrophobic clefts in the surface of the ClpP P-ring.

Supplementary information

Supplementary Information

Supplementary Tables: Table 1. Summary information on peptides identified by mass spectrometry. Table 2. Summary information on sequences solved in the structure. In column 4, the numbering is based on the mature protein, except for ClpP1C (based on ClpP1H precursor). Sequences solved up to the N or C termini are in bold letters. Table 3. Cryo-EM data collection and refinement statistics.

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Supplementary Data

Supplementary Data 1.

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 2

Unprocessed gels.

Source Data Extended Data Fig. 2

Unprocessed gels.

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Wang, N., Wang, Y., Zhao, Q. et al. The cryo-EM structure of the chloroplast ClpP complex. Nat. Plants 7, 1505–1515 (2021). https://doi.org/10.1038/s41477-021-01020-x

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