1. Structural Biology and Molecular Biophysics
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Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates

  1. Xue Fei
  2. Tristan A Bell
  3. Sarah R Barkow
  4. Tania A Baker
  5. Robert T Sauer  Is a corresponding author
  1. Departments of Biology, Massachusetts Institute of Technology, United States
  2. Chemistry, Massachusetts Institute of Technology, United States
Research Article
Cite this article as: eLife 2020;9:e61496 doi: 10.7554/eLife.61496
5 figures, 4 videos, 3 tables and 1 additional file

Figures

Figure 1 with 3 supplements
ClpXP complexes with ssrA degrons.

(A) Side and top views of the composite cryo-EM density from the recognition complex. (B) The upper portion of the panel shows transparent density for the ssrA degron (stick representation) in the recognition and intermediate complexes; the lower portion shows the offset positions of the ssrA degron (space-filling representation) in the channel of ClpX (cartoon representation) in both complexes after removal of subunit F. In this and all subsequent figures, ClpX is colored blue, green, or purple; ClpP is yellow; and substrate is orange/gold.

Figure 1—figure supplement 1
Cryo-EM data and strategy.

Data processing workflow. EM images contained singly capped complexes (one ClpX hexamer bound to one ClpP tetradecamer) or doubly capped complexes (two ClpX hexamers bound to one ClpP tetradecamer). Singly capped complex (red boxes) were selected for further refinement and classification. The recognition and intermediate complexes accounted for 92% of all of the singly capped complexes.

Figure 1—figure supplement 2
Cryo-EM data validation.

(A) Density maps of ClpP, the recognition complex, and the intermediate complex are colored by local resolution. (B) Fourier Shell Correlation (FSC) plots of half maps (blue) or model-map (red). Dashed lines show a cut-off value of 0.143 (blue) or 0.5 (red). Resolutions reported are for a half map FSC of 0.143. (C) Euler-angle orientation distribution for the recognition and intermediate complexes.

Figure 1—figure supplement 3
Representative density.

Density for portions of the ClpX component of the recognition complex is shown in panel (A); for the ClpX component of the intermediate complex in panel (B); and for ClpP in panel (C).

Figure 2 with 2 supplements
ClpX-degron interactions.

(A) Positions of the RKH loop (blue), pore-1 loop (red), and pore-2 loop (green) in subunit A of the ClpX hexamer in the recognition complex relative to the positions of the ssrA degron (orange/gold). (B) Cutaway views of the recognition complex (left) and intermediate complex (right). In the recognition complex, the pore-2 loop of ClpX subunit A (denoted by red arrows) blocks the axial pore. The pore is open in the intermediate complex. (C) Key ClpX residues (blue or purple) and the C-terminal segment of the ssrA degron (gold) in the recognition complex are shown in stick representation. Dashed lines indicate hydrogen bonds. (D) ClpX-degron contacts in the recognition and intermediate complexes. ClpX or degron residues are shown in stick representation with semi-transparent density. (E) Subunit nucleotide state and degron/pore-loop interactions in the recognition and intermediate complexes.

Figure 2—figure supplement 1
Detailed contacts between ClpX and the ssrA degron.

(A) Schematic representation of recognition-complex contacts between ClpX side chains and the ssrA degron prepared using LigPlot (Wallace et al., 1995). Dashed lines indicate hydrogen bonds. Hydrophobic and van der Waal’s contacts are indicated by arcs with spokes. (B) LigPlot schematic of ClpX-degron contacts in the intermediate complex. (C, D) Surface area buried in the recognition or intermediate complexes between the ssrA degron and ClpX residues (X-axis) for different ClpX subunits (Y-axis).

Figure 2—figure supplement 2
Nucleotide density in subunits of the recognition complex (top) and the intermediate complex (bottom).
Mutations affecting recognition-complex contacts increase KM for ClpXP degradation.

(A) Steady-state KM (top) and Vmax (bottom) parameters (means ± SD; n = 3) for wild-type ClpXP degradation of synthetic peptide substrates containing an aminobenzoic-acid (ABZ) fluorophore and nitrotyrosine (YNO2) quencher (wild-type sequence ABZ-FAPHMALVPYNO2GYGGKKLAANDENYALAA; gray bar) with substitutions for the penultimate residue (green bars), C-terminal residue (dark red bars), or α-carboxylate (red bar). The statistical significance of KM or Vmax values relative to the wild-type Ala-Ala-COO values was calculated using Student's two-tailed t-test (ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001). (B) Degradation of GFP-ssrA by ClpX∆N/ClpP and variants. Data are means ± SD (n = 3 to 6), and lines are fits to the Michaelis-Menten equation. Fitted KM values (µM) were 1.3 ± 0.2 (wild type), 6.3 ± 1.7 (T199S), and 89 ± 34 µM (T199V). Fitted Vmax values (min−1 ClpX−1) were 2.0 ± 0.1 (wild type), 3.1 ± 0.2 (T199S), and 3.7 ± 2.0 (T199V). For the T199A, V202A, and H230A variants, unconstrained fits had huge error limits, and fits with Vmax constrained to less than or equal to 4 min−1 ClpX−1 gave KM values from 140 to 240 µM.

Degradation dependence on degron length.

(A) Cartoon of GFP (pdb 1EMA; Ormö et al., 1996) with degron tails of 3, 5, 7, 9, and 11 residues. (B) Plots of steady-state ClpX∆N/ClpP degradation rates (means ± SD; n = 3) as a function of substrate concentration. Lines are fits to the Michaelis-Menten equation. Fitted KM and Vmax values represent averages of three independent determinations ± SD. (C) Model of GFP-yalaa bound to ClpXP (cartoon and semi-transparent surface representation) created by aligning the yalaa of GFP to the same sequence in the recognition complex and then minimizing clashes both manually and computationally. (D) Close-up view of the ClpX-GFP-yalaa model (subunits E and F removed), corresponding to the dashed rectangle in panel C, with the yalaa shown in ball-and-stick representation. (E) Cartoon in which one power stroke unfolds GFP by translocating the yalaa degron six residues deeper into the channel of ClpX.

Models for substrate recognition, engagement, and unfolding by ClpXP.

Top. A substrate with a relatively long degron (~20 residues) is recognized and subsequent ATP-dependent power strokes then move the degron deeper into the ClpX channel in the intermediate complex, and then the engaged complex, from which unfolding occurs. Bottom. A substrate with a short degron (~5 residues) forms a recognition complex that is engaged and can therefore carry out direct ATP-dependent unfolding.

Videos

Video 1
Interaction of the ssrA degron with ClpX in the recognition complex.

The ssrA tag is contacted by pore loops located at the top of the axial channel of ClpX. EM density is shown as a transparent surface. Hydrogen bonds between pore loops and the ssrA degron are shown as dashed lines.

Video 2
Interaction of the ssrA degron with ClpX in the intermediate complex.

The pore-1 and pore-2 loops from different subunits of ClpX interact with every two residues of the ssrA degron, as observed in other structures of ClpXP and related AAA+ proteins. EM density is shown as a transparent surface. Hydrogen bonds between pore loops and the ssrA degron are shown as dashed lines.

Video 3
Model of the interaction between GFP-yalaa and the ClpX portion of the recognition complex.

GFP with a five-residue yalaa degron docks snuggly and without major clashes with the ClpX ring.

Video 4
Side and top views of a morph between the recognition and intermediate complexes with one ClpX subunit removed for clarity.

This morph was generated by aligning subunit A in the recognition complex spiral with subunit F in the intermediate complex spiral after superimposing the ClpP portions of the two structures. In the morph, the purple subunit and ssrA degron move 25 Å or six residues deeper into the axial channel toward ClpP.

Tables

Table 1
Cryo-EM data collection, processing, model building, and validation statistics.
NameClpPClpXP-ssrA
Recognition
complex
ClpXP-ssrA
Intermediate
complex
PDB ID6WR26WRF6WSG
EMDB IDEMD-21875EMD-21882EMD-21892
Data collection/processing
MicroscopeTalos Arctica
CameraK3
Magnification45,000X
Voltage (kV)200
Total electron dose (e-2)53
Defocus range (µm)−1.2 to −2.5
Pixel size (Å)0.435
Micrographs collected4525
Final particles344069139817130240
SymmetryC1C1C1
Resolution Å (FSC 0.143)2.83.13.2
Model composition
Non-hydrogen atoms21,31026,83525,932
Protein residues272934503334
Ligands065
Refinement
Map-model CC0.820.770.74
RMSD bond lengths (Å)0.0150.0030.011
RMSD bond angles (degrees)1.160.731.13
Validation
MolProbity score0.890.940.96
Clash score1.51.91.7
C-beta deviations000
Rotamer outliers (%)00.070
Ramachandran favored (%)98.99999.3
Ramachandran disallowed (%)000
Table 2
Comparisons between high-resolution ClpXP structures.

(A). RMSDs between Cα positions. (B). Nucleotides bound in different subunits of high-resolution ClpXP structures. Gray shading indicates subunits that always contain ATP or ATPγS.

Table 2A
PDB IDNameReferenceRMSD (Å)RMSD (Å)
Recognition complexIntermediate complex
6WSGIntermediate complexThis paper2.80.0
6PP8Class 1Fei et al., 20202.01.9
6PP7Class 2Fei et al., 20202.81.3
6PP6Class 3Fei et al., 20201.42.7
6PP5Class 4Fei et al., 20201.42.9
6VFSConformation ARipstein et al., 20203.02.6
6VFXConformation BRipstein et al., 20201.63.0
Table 2B
PDB IDNameClpX subunitReference
ABCDEF
6WRFRecognition complexATPATPATPATPATPADPThis paper
6WSGIntermediate complex-ATPATPATPATPATPThis paper
6PP8Class 1ATPATPATPATPATPADPFei et al., 2020
6PP7Class 2ADPATPATPATPATPATPFei et al., 2020
6PP6Class 3ATPATPATPATPATPADPFei et al., 2020
6PP5Class 4ATPATPATPATPATPADPFei et al., 2020
6VFSConformation AADPATPATPATPATPADPRipstein et al., 2020
6VFXConformation BATPATPATPATPATPADPRipstein et al., 2020
Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain background (Escherichia coli)ER2566NEB1312Chemically competent cells
Gene (Escherichia coli)clpXE. coli (strain K12) EXPASYUniProtKB- P0A6H1
Gene (Escherichia coli)clpPE. coli (strain K12) EXPASYUniProtKB- P0A6G7
Recombinant DNA reagentpT7 ClpX (plasmid)Kim et al., 2000N-terminally His6-tagged ClpX for overexpression
Recombinant DNA reagentpT7 ClpXΔN(plasmid)Martin et al., 2005N-terminally His6-tagged ClpXΔN(residues 62–424) for overexpression
Recombinant DNA reagentpT7-ClpXΔN-T199A (plasmid)This paper, Material and methodsClpXΔN (residues 62–424) T199A mutant, can be obtained from the Sauer lab
Recombinant DNA reagentpT7-ClpXΔN-T199S (plasmid)This paper, Material and methodsClpXΔN (residues 62–424) T199S mutant, can be obtained from the Sauer lab
Recombinant DNA reagentpT7-ClpXΔN-T199V (plasmid)This paper, Material and methodsClpXΔN (residues 62–424) T199V mutant, can be obtained from the Sauer lab
Recombinant DNA reagentpT7-ClpXΔN-V202A (plasmid)This paper, Material and methodsClpXΔN (residues 62–424) V202A mutant, can be obtained from the Sauer lab
Recombinant DNA reagentpT7-ClpXΔN-H230A (plasmid)This paper, Material and methodsClpXΔN (residues 62–424) H230A mutant, can be obtained from the Sauer lab
Recombinant DNA reagentPACYC-ClpXΔN6-TEV-cHis6 (plasmid)Martin et al., 2005ClpX expression, can be obtained from the Sauer lab
Recombinant DNA reagentpT7 ClpP (plasmid)Kim et al., 2000C-terminally His6-tagged ClpP for overexpression
Recombinant DNA reagentpT7-GFP-ssrA
(plasmid)
Kim et al., 2000Expresses fluorescent substrate for degradation assays, can be obtained from the Sauer lab
Recombinant DNA reagentpT7 GFP LAA (plasmid)This paperN-terminally His6-tagged GFP (1-229) substrates with a LAA C-terminal tail, for overexpression.
Recombinant DNA reagentpT7 GFP YALAA (plasmid)This paperN-terminally His6-tagged GFP (1-229) substrates with a YALAA C-terminal tail, for overexpression.
Recombinant DNA reagentpT7 GFP ENYALAA (plasmid)This paperN-terminally His6-tagged GFP (1-229) substrates with a ENYALAA C-terminal tail, for overexpression.
Recombinant DNA reagentpT7 GFP NDENYALAA (plasmid)This paperN-terminally His6-tagged GFP (1-229) substrates with a NDENYALAA C-terminal tail, for overexpression.
Recombinant DNA reagentpT7 GFP AANDENYALAA (plasmid)This paperN-terminally His6-tagged GFP (1-229) substrates with a AANDENYALAA C-terminal tail, for overexpression.
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALAA-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALGA-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALIA-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALFA-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALDA-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALKA-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALAG-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALAI-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALAF-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALAD-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALAK-COO-This paperFluorescent peptide, for degradation assays
Peptide, recombinant proteinABZ-FAPHMALVPYNO2GYGGKKLAANDENYALAA-CONH2This paperFluorescent peptide, for degradation assays
Software, algorithmRelionZivanov et al., 2018RRID:SCR_016274EM reconstruction software
Software, algorithmUCSF ChimeraPettersen et al., 2004RRID:SCR_004097Molecularvisualizationsoftware
Software, algorithmUCSF ChimeraXGoddard et al., 2018RRID:SCR_015872Molecularvisualizationsoftware
Software, algorithmPhenixAdams et al., 2010RRID:SCR_014224Structure refinement software
Software, algorithmMolProbityWilliams et al., 2018RRID:SCR_014226Protein modelevaluationsoftware
Software, algorithmPyMOLSchrödinger, LLC.RRID:SCR_000305Molecularvisualizationsoftware
Software, algorithmCootEmsley and Cowtan, 2004RRID:SCR_014222Protein modelbuildingsoftware
Software, algorithmCtffindRohou and Grigorieff, 2015RRID:SCR_016732EM image analysis software
Software, algorithmPISA'Protein interfaces, surfaces and assemblies' service
PISA at the European Bioinformatics Institute (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html)
RRID:SCR_015749Protein modelanalysis software

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