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Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and proteolysis

Abstract

The ClpAP complex is a conserved bacterial protease that unfolds and degrades proteins targeted for destruction. The ClpA double-ring hexamer powers substrate unfolding and translocation into the ClpP proteolytic chamber. Here, we determined high-resolution structures of wild-type Escherichia coli ClpAP undergoing active substrate unfolding and proteolysis. A spiral of pore loop–substrate contacts spans both ClpA AAA+ domains. Protomers at the spiral seam undergo nucleotide-specific rearrangements, supporting substrate translocation. IGL loops extend flexibly to bind the planar, heptameric ClpP surface with the empty, symmetry-mismatched IGL pocket maintained at the seam. Three different structures identify a binding-pocket switch by the IGL loop of the lowest positioned protomer, involving release and re-engagement with the clockwise pocket. This switch is coupled to a ClpA rotation and a network of conformational changes across the seam, suggesting that ClpA can rotate around the ClpP apical surface during processive steps of translocation and proteolysis.

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Fig. 1: Architecture of the substrate-bound ClpAP complex.
Fig. 2: Three distinct structures of ClpAP showing IGL loop rearrangement.
Fig. 3: IGL loop interactions and conformational flexibility.
Fig. 4: Structure of ClpP and NT gating loops.
Fig. 5: ClpA pore loop–substrate contacts and translocation states.
Fig. 6: Nucleotide states and ClpA rotation model for processive unfolding and proteolysis.

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Data availability

ClpAP cryo-EM maps and atomic coordinates have been deposited in the EMDB and PDB with accession codes EMD-21519 and PDB 6W1Z for ClpAPEng1; EMD-21520 and PDB 6W20 for ClpAPDis; EMD-21521 and PDB 6W21 for ClpAPEng2; EMD-21522 and PDB 6W22 for ClpAPEng1 focus; EMD-21523 and PDB 6W23 for ClpAPDis focus; EMD-21524 and PDB 6W24 for ClpAPEng2 focus; EMD-20851 and PDB 6UQO for ATPγS-ClpAPEng; and EMD-20845 and PDB 6UQE for ATPγS-ClpAPDis. Uncropped images for Extended Data Fig. 1a,c are provided as Source Data online.

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Acknowledgements

We thank K. Mack, Z. March, R. Cupo, T. Pospiech and J. Braxton for feedback on the manuscript. We thank the UCSF BACEM Facility for assistance with data collection. This work was supported by an Alzheimer’s Association Research Fellowship (to J.B.L.), a GAANN fellowship (to A.N.R.), NSF grant no. MCB-1412624 (to A.L.L.) and NIH grant nos. R01GM099836 (to J.S.) and R01GM110001 (to D.R.S.).

Author information

Authors and Affiliations

Authors

Contributions

K.E.L. and A.N.R. carried out all experiments, refinement and modeling procedures for structure determination; developed figures; and wrote and edited the manuscript. E.T. operated the Krios microscope and helped with data collection. J.B.L. performed biochemical substrate-binding experiments. N.W.S. expressed and purified protein components. A.C.T. performed degradation assays. A.L.L. and J.S. wrote and edited the manuscript. D.R.S. designed and supervised the project and wrote and edited the manuscript.

Corresponding author

Correspondence to Daniel R. Southworth.

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The authors declare no competing interests.

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Peer review information Inês 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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Extended data

Extended Data Fig. 1 ClpAP complex formation with RepA(1-25)-GFP and cryoEM data analysis.

a RepA1–25-GFP degradation assay in the presence of either ATPγS or ATP along with ClpA and ClpP. The assay was performed at 20°. Arrow represents RepA degradation product. b Size exclusion chromatography (SEC) trace of the components and formed ClpAP complex following incubation with RepA1–25-GFP and ATPyS. The 280 absorbance traces are shown for ClpA alone (red, dashed), ClpA with RepA1–25-GFP (red, solid), ClpAP alone (black, dashed) and ClpAP with RepA1–25-GFP (black, solid). c RepA1–25-GFP degradation assay in the presence of ATPγS with both ClpP WT and ClpP_S98A. ATP was spiked into the reaction at 10 mM after the initial complex formation for 15 min was completed with ATPγS. The zero-time point is before spiking ATP into the reaction. The assay was performed at 20°. d Reference-free 2D class averages of ClpAP bound to RepA1–25-GFP. The scale bar equals 125 Å. e Gold standard FSC-curves for the final refinement of ClpAPEng1(red), ClpAPDis(cyan), ClpAPEng2(black) of the ClpAP-RepA(1-25)-GFP complex. f 3D classification scheme used to identify the two different states in the ClpAP-RepA1–25-GFP dataset. Green asterisk represents the classes in which the particles were pooled together for further classification and refinement. The local resolution map of ClpAPEng-1 (g), ClpAPDis (h) and ClpAPEng-2 (i). j Low-pass filtered map showing globular density docked with GFP (PDB 1GFL) and additional N-terminal ClpA density (NTD). k Map vs. Model FSC of ClpAPEng1(red), ClpAPDis(cyan), ClpAPEng2 (black) of the ClpAP-RepA(1-25)-GFP complex following atomic modeling in Rosetta. Uncropped gel images are available as source data online.

Source data

Extended Data Fig. 2 Difference maps of the ClpAP interface.

Difference maps of the cryo-EM maps of a ClpAPEng1 vs. ClpAPDis and ClpAPEng-2, b ClpAPDis vs. ClpAPEng-1 and ClpAPEng-2, c ClpAPEng-2 vs. ClpAPDis and ClpAPEng-1. The IGL pockets are encompassed by red circle, open pocket (dashed) and occupied pocket (solid). Schematic (right) shows occupancy of the ClpA IGL-loops (circles, colored and numbered by protomer) around the ClpA hexamer, with the empty IGL pockets (white circles) and ClpA protomers indicated (letters) for the different states. Asterisk represents the IGL-loop that is engaging in that state.

Extended Data Fig. 3 ATPγS-ClpAP cryoEM data analysis.

a Reference-free 2D class averages of ClpAP-γS bound to RepA1–25-GFP. The scale bar equals 125 Å. b Gold standard FSC-curves for the final refinement of ATPγS-ClpAPEng (blue) and ATPγS-ClpAPDis (red) of the ClpAP-RepA(1-25)-GFP complex. ATPγS-ClpAPEng1 (c) and ATPγS-ClpAPDis (d) cryo-EM maps showing degree offset (arrow) of the ClpA channel axis (solid line) and substrate position (yellow density) compared to the ClpP pore and proteolytic chamber (dashed line). Schematic (below,left) shows occupancy of the ClpA IGL-loops (circles, colored and numbered by protomer) around the ClpA hexamer, with the empty IGL pockets (white circles) and ClpA protomers indicated (letters) for the different states. e 3D classification scheme used to identify the two different states in the ATPγS-ClpAP-RepA1–25-GFP dataset. Dotted boxes represent the classes in which the particles were pooled together for further classification and refinement. The maps for ClpAPEng (red) and ClpAPDis (yellow) are colored accordingly. Map vs. Model FSC of ATPγS-ClpAPEng(f) and ATPγS-ClpAPDis(g) following atomic modeling in Rosetta.

Extended Data Fig. 4 Comparison of IGL loops between the different states.

a EM map and model of the IGL-loop in the hydrophobic pocket of P1 (top), P2-P4 (middle, top), P5 (middle, bottom) and P6 (bottom) for ClpAPEng1(left), ClpAPDis (middle) and ClpAPEng2 (right). b Overlay of IGL-loops of ClpAPEng1 (colored by protomer) vs. ClpAPDis (black) vs. ClpAPEng2 (grey) laid out after alignment to the residues (638-649) above the IGL-loop. The dotted loop in P1 represents the missing loop in ClpAPDis and ClpAPEng-2.

Extended Data Fig. 5 Single capped ClpAP structure and ClpP N-terminal loop interactions.

a Map of the ClpP N-terminal gating loops and the model for ClpA with substrate for ClpAPDis (b) ClpAPEng. Map and model view of ClpP residues E14 and R15 (c) and E8 and K25 (d). e Gold standard FSC curve and (f) 2D reference-free class averages of the single capped ClpAP structure.

Extended Data Fig. 6 Particle Subtraction and Focus Refinement of ClpAPEng1, ClpAPEng2 and ClpAPDis.

a EM map with mask (grey) used for particle subtraction of ClpA. Red dot represents the point in which particles were shifted to. b Gold standard FSC curve of both focus maps for ClpAPEng1 (red), ClpAPDis (cyan), and ClpAPEng2 (black). The local resolution map of ClpAPEng1 (c), ClpAPDis (d) and ClpAPEng2 (e). f EM map and model of each Tyr-containing pore loop in ClpAPEng1 for both D1 (top) and D2 (bottom), the substrate channel density is colored yellow. g EM map and model of each Tyr-containing pore loop in P5 for ClpAPEng1 (left), ClpAPDis (middle), and ClpAPEng2 (right) for both D1 (top) and D2 (bottom), the substrate channel density is colored yellow. The distance between the Tyr and the substrate is represented by dotted line. h EM map and model of ClpAPEng1 (colored by protomer) with the D2 secondary pore loops residues interacting with substrate. i ClpAPEng1 EM map colored by protomer with D2 secondary pore loops (red) and ClpP NTD-loops (green). j Overlay of the seam protomers P5 (left), P1 (middle), and P6 (right) for ClpAPEng1 (grey) and ClpAPEng2 (colored) showing conformational shifts (arrows) supporting translocation step.

Extended Data Fig. 7 Nucleotide States of ClpAPEng1, ClpAPEng2 and ClpAPDis.

a Difference map density for P4 D1 and D2 ATP with Arg finger residues displayed in green. There are no differences between P3 and P4, therefore P3 ATP density is not shown. b Difference map density for P1, P2, P5 and P6 for both D1 and D2 and Arg finger residues colored green.

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Reporting Summary

Supplementary Video 1

Pivot of ClpA on ClpP between the engaged and disengaged states.

Supplementary Video 2

The IGL loop plasticity between the engaged and disengaged states.

Supplementary Video 3

The substrate contacts of the Tyr-containing pore loops in both the engaged and disengaged states.

Supplementary Video 4

Movement of seam protomers (P1, P5 and P6) between the engaged and disengaged states. The movie depicts a morph between the Engaged-1, Disengaged and Engaged-2 states and the two models are aligned to the stationary protomers (P2, P3 and P4). The P1 IGL loop is not displayed in the movie.

Supplementary Video 5

Overall dynamic translocation mechanism of ClpAP involving substrate release and IGL loop binding and re-binding at the spiral seam.

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Lopez, K.E., Rizo, A.N., Tse, E. et al. Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and proteolysis. Nat Struct Mol Biol 27, 406–416 (2020). https://doi.org/10.1038/s41594-020-0409-5

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