Cryo-EM structure of the ClpXP protein degradation machinery

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Abstract

The ClpXP machinery is a two-component protease complex that performs targeted protein degradation in bacteria and mitochondria. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP, where they are degraded. Formation of the complex involves a symmetry mismatch, because hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamer-hexamer binding interface and provide novel insight into the ClpX-ClpP cross-talk and activation mechanism. Comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of the complex mode of action of ClpXP and provides a structural framework for future pharmacological applications.

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Fig. 1: LmClpXP1−2 forms flexible dimers via the ZBDs.
Fig. 2: Cryo-EM structure of the ClpXP1−2 protein degradation machinery.
Fig. 3: Symmetry mismatch between ClpP1−2 and ClpX.
Fig. 4: HDX-MS analysis of ClpXP1−2 complex formation.
Fig. 5: Role of the ClpP2 C terminus in ClpXP1−2 binding.
Fig. 6: Comparison of ClpX-bound ClpP1−2 with available structures of active and inactive ClpP.
Fig. 7: ClpX binds to ClpP in a similar manner to ADEP but does not induce ClpP pore widening.

Data availability

The cryo-EM map of LmClpXP1−2 has been deposited at the EMDB with the accession code EMD-10162. The corresponding molecular models of LmClpX and LmClpP1−2 have been deposited at the wwPDB with accession codes PDB 6SFW and PDB 6SFX, respectively. Source data for Fig. 4, Fig. 5b,c, Supplementary Fig. 1a and Supplementary Fig. 6 are available online. All data used in this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank O. Hofnagel for assistance in electron microscopy and Dr. M. Lakemeyer for building ClpX homology models. We are grateful to Dr. M. Haslbeck, G. M. Feind and F. Rührnößl for HDX-MS measurements. This work was supported by the Max Planck Society (to S.R.), the European Research Council (FP7/2007-2013) (grant no. 615984) (to S.R.) and the Deutsche Forschungsgemeinschaft (SFB1035) (to S.A.S).

Author information

S.A.S. and S.R. designed the study. C.G. screened and optimized samples, prepared cryo-EM grids and processed and analyzed cryo-EM data. D.B. cloned, overexpressed and purified proteins, optimized sample preparation, conducted activity assays and gel filtration measurements and analyzed HDX-MS data. C.G. and F.M. built atomic models. C.G. and D.B. prepared figures, C.G., D.B., S.A.S. and S.R. wrote the manuscript. All authors discussed the results.

Correspondence to Stephan A. Sieber or Stefan Raunser.

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The authors declare no competing interests.

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Peer review information Ines Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary Figure 1 EM analysis of the ClpXP1–2 dimer.

a) Size exclusion chromatography of ClpX and ClpP1–2 mixtures on a Superose 6 increase 10/300 column. For the EM studies, a sample at 12 mL was taken. Note that the (ClpXP1–2)2 peak is absent with ClpXΔZBD. Source data are available online. b) SDS-PAGE of the isolated (ClpXP1–2)2 complex. cd) Subarea of a negative stain EM micrograph of the isolated (ClpXP1–2)2 prior (c) and after (d) crosslinking; Scale bar, 200 nm e) Fourier Shell Correlation (FSC) between two independently refined half maps f) Low resolution cryo-EM density of the ClpXP1–2-dimer g) 3D clustering of the ClpXP1–2-dimer dataset. Source data

Supplementary Figure 2 Cryo-EM analysis of ClpXP1–2.

a) Subarea of a typical low-dose cryo-EM micrograph of ClpXP1–2. ClpXP1–2 particles were selected and extracted from ClpXP1–2-ClpXP1–2 dimers using crYOLO and highlighted in red boxes. Scale bar, 100 nm b) Representative reference-free 2D class averages of ClpXP1–2. Scale bar, 25 nm c) Fourier Shell Correlation (FSC) between two independently refined half maps d) Side and cut-off view of the density map colored according to the local resolution e) Orientation distribution of the particles used in the final refinement round f-g) Superposition of segments of the molecular model of ClpP (f) and ClpX (g) with the cryo-EM density (transparent surface).

Supplementary Figure 3 ClpP-bound ClpX subunits adopt a nucleotide-loadable conformation.

Molecular models of the six ClpX subunits are shown as ribbon diagrams, with the large domain highlighted in orange and the small domain in red. Note the high similarity between the ClpX subunits, except the arrangement of their IGF-loops. The gray arrow indicates the IGF-loop of subunit Q that adopts an ‘extended’ conformation. The inset shows nucleotide-loadable (L; upper image) and unloadable (U; lower image) subunits of ATPΥS-bound EcClpX (PDB 4I81). Structural comparison of the six ClpX subunits with the L and U subunit of EcClpX (note the respective RMSD values of the Cα atoms) indicate that all subunits of ClpP-bound ClpX adopt a loadable conformation.

Supplementary Figure 4 HDX-MS of the ClpXP1–2 complex.

Changes in deuterium uptake after complex formation are mapped on the amino acid sequence of a) ClpX, b) ClpP1 and c) ClpP2 for the respective exposure times. Increased deuterium uptake upon complex formation is shown in red, decreased deuterium uptake is depicted in blue. Dark gray represents no coverage. Please refer to the Methods section for the calculation of the relative deuterium uptake values. Averages of two independent measurements are shown.

Supplementary Figure 5 Cryo-EM density for the IGF-loops interfaces and the ClpP2 catalytic active site.

a) Densities for the six IGF-loops interactions are shown with the corresponding atomic models. ClpX and ClpP2 densities are shown as gray and green transparent isosurface, respectively. b) Superposition of the catalytic residues S98 (S98A), H123 and D172 in ClpX-bound LmClpP1-S98A/P2-S98A (cryo-EM) (extended active state) and SaClpP (compact inactive state) (PDB 4EMM), shown with the cryo-EM density. The catalytic residues of ClpX-bound LmClpP1–P2 adopt the active conformation.

Supplementary Figure 6 Activity assays of ClpX and ClpP2 mutants of the IGF-loop/hydrophobic groove interface.

a) Peptidase activity of ClpP1–2 with respective ClpP2 mutants (0.71 μM (ClpP1–2)14, 100 μM Ac-Ala-hArg-2-Aoc-ACC). b) ATPase activity of ClpX mutants (0.33 μM ClpX6, 20 mM ATP). c) Protease activity of ClpXP1–2 with ClpP2 and ClpX mutants (0.2 μM (ClpP1–2)14, 0.4 μM ClpX6, 0.8 μM GFP-SsrA). Data are normalized to the wild type as 100% (n = 6, black lines denote means). Source data for graphs in a-c are available online. d) Mapping of the ClpP2 and ClpX mutations on the protein structure. Mutation sites are shown with red sticks. Source data

Supplementary Figure 7 The N-terminal loops of ClpX-bound ClpP2 subunits adopt the “up” conformation.

Cryo-EM density map (mesh) with the molecular model highlighting the N-terminal domain of the seven ClpP subunits. Residues 8-17 are not resolved, but the fragmented cryo-EM density indicates that all flexible N-terminal loops adopt the “up” conformation (indicated by dashed lines). For better comparison, the molecular model of a subunit of EcClpP (PDB 1YG6) with the N-terminus in the “down” conformation (orange) is also shown.

Supplementary Figure 8 Possible interactions between pore-2 loops of ClpX with the N-termini of ClpP.

a-f) Cryo-EM density map with the molecular model, highlighting the interaction area between the pore-2 loops of ClpX and the N-termini of ClpP. The six pore-2 loops of ClpX and residues 7-16 of the N-termini of seven subunits of ClpP2 are not resolved. Possible arrangements of these regions are indicated by dashed lines, based on their anchor points and number of residues. Note that the pore-2 loops of chains Q and P point into a cleft formed by three ClpP N-termini (b,c). This topological analysis also suggests that the pore-2 loops of chains O and T (a,f) do not show any interactions with the N-termini of ClpP. However, an unusual stretched conformation of these pore-2 loops towards ClpP cannot be excluded. Pore-2 loop of chain S is positioned in direct proximity to the N-terminus of chain N (c) whereas the pore-2-loop of chain R is positioned between two ClpP N-termini (M and N) (e).

Supplementary Figure 9 Alignment of ClpP sequences.

Mt = Mycobacterium tuberculosis (ClpP2), Pa = Pseudomonas aeruginosa (ClpP1), Cd = Clostridium difficile (ClpP1 and ClpP2), Ec = Escherichia coli, Lm = Listeria monocytogenes (ClpP2), Bs = Bacillus subtilis, Sa = Staphylococcus aureus.

Supplementary information

Supplementary Information

Supplementary Figs. 1–9 and Table 1

Reporting Summary

Supplementary Video 1

Flexibility of ClpXP1/2 dimers in 2D.

Supplementary Video 2

Structural comparison between ClpX-bound and ADEP-bound ClpP. The video shows a simple linear interpolation between ClpX-bound ClpP1/2 (cryo-EM) (extended active conformation) and the available crystal structure of B. subtilis ADEP2-bound ClpP (PDB 3KTK41) (extended active open conformation), first along the ClpP1 and finally along the ClpP2 face (see Fig. 6c). Note the widening of the pore in the ADEP-bound structure.

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