The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase

  1. Valentin Bohl
  2. Nele Merret Hollmann
  3. Tobias Melzer
  4. Panagiotis Katikaridis
  5. Lena Meins
  6. Bernd Simon
  7. Dirk Flemming
  8. Irmgard Sinning
  9. Janosch Hennig
  10. Axel Mogk  Is a corresponding author
  1. Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Germany
  2. Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL) Heidelberg, Germany
  3. Heidelberg University Biochemistry Center (BZH), Germany
  4. Chair of Biochemistry IV, Biophysical Chemistry, University of Bayreuth, Germany
8 figures and 3 additional files

Figures

Figure 1 with 2 supplements
ClpL is an autonomous disaggregase.

(A) Domain organizations of ClpL, ClpG, ClpB. All AAA+ proteins consist of two AAA domains (AAA1, AAA2), a coiled-coil middle domain (M) and diverse N-terminal domains (N). ClpG additionally harbors …

Figure 1—figure supplement 1
Sequence alignment of Escherichia coli ClpB, S. aureus ClpC, P. aeruginosa ClpGGI, and L. monocytogenes ClpL.

The domain organization is indicated. Similar and identical residues are highlighted in light and dark blue.

Figure 1—figure supplement 2
ClpL is a potent, stand-alone disaggregase.

(A) Relative luciferase disaggregation activities (% refolded luciferase/min) of ClpL in the presence of 2 mM ATP (and a regeneration mix), 2 mM ADP and without nucleotide were determined. The …

Figure 2 with 1 supplement
Specific molecular features separate ClpL from ClpB/DnaK.

(A) Incubation of luciferase-YFP at 46°C only leads to unfolding of the luciferase moiety and the formation of mixed aggregates including folded YFP. Unfolding of YFP during disaggregation of …

Figure 2—figure supplement 1
Unfolding of DnaK at high temperatures is reversible.

Relative luciferase disaggregation activities (% refolded luciferase/min) of ClpL and L. monocytogenes (Lm) ClpB/KJE were determined. ClpL or DnaK were incubated for 30 min at the indicated …

Figure 3 with 2 supplements
The ClpL N-terminal domain (NTD) is essential for disaggregation activity.

(A) Disaggregation activities of ClpL and ΔN-ClpL toward aggregated luciferase and malate dehydrogenase (MDH) (each: % regain enzymatic activity/min) were determined. The activity of ClpL was set to …

Figure 3—figure supplement 1
ClpL disaggregation activity relies on ATP-fueled substrate threading.

(A) Domain organization of ClpL. Pore loop (Y170, Y504) and Walker B (E197, E530) residues of AAA1 and AAA2 domains are indicated. (B) ClpL ATPase activities were determined at 0.125 and 1 μM …

Figure 3—figure supplement 2
The ClpL N-terminal domain (NTD) is crucial for disaggregation activity.

(A) E. coli dnaK103 cells harboring plasmids for IPTG-controlled expression of indicated disaggregases were grown at 30°C to mid-logarithmic growth phase and shifted to 49°C. Serial dilutions of …

Figure 3—figure supplement 2—source data 1

Source data include non-cropped and non-processed images of SDS-gels and western blots.

https://cdn.elifesciences.org/articles/92746/elife-92746-fig3-figsupp2-data1-v2.zip
Figure 3—figure supplement 2—source data 2

Source data include non-cropped and non-processed images of SDS-gels and western blots and indicate sections and loading schemata of the respective figure supplement.

https://cdn.elifesciences.org/articles/92746/elife-92746-fig3-figsupp2-data2-v2.zip
Figure 4 with 2 supplements
Molecular basis of ClpL N-terminal domain (NTD) binding to protein aggregates.

(A) AlphaFold2 model of ClpL NTD. The color code depicts the calculated confidence of the prediction (pLDDT). Residues that potentially participate in the formation of small hydrophobic cores (α1/2 …

Figure 4—figure supplement 1
Structural analysis of ClpL N-terminal domain (NTD).

(A) 1H,15N-HSQC of ClpL NTD with resonance assignment labeled at each peak. Several residues exhibit dispersed peak position, indicative of folding. (B) 1H,1H-2D NOESY in D2O of ClpL NTD confirming …

Figure 4—figure supplement 2
Mutant analysis of ClpL N-terminal domain (NTD).

(A/D) ATPase activities of indicated ClpL mutants determined at 0.125 and 1 μM protein concentration. The ATPase activities of ClpL wild-type (WT) were set to 1. (B/C) Luciferase and malate …

Figure 5 with 1 supplement
Multiple ClpL N-terminal domains (NTDs) are required for disaggregation activity.

(A) Varying the ratio of LN-ClpB* and ΔN-ClpB* leads to formation of mixed hexamers with diverse numbers of NTDs. (B) Luciferase disaggregation activities (% refolded luciferase/min) of mixed LN-ClpB…

Figure 5—figure supplement 1
Multiple ClpL N-terminal domains (NTDs) are required for disaggregation activity.

(A) Relative luciferase disaggregation activities (% refolded luciferase/min) of indicated LN-ClpB* disaggregase mixtures were determined (DWB: ATPase-deficient LN-ClpB*-E279A/E678A, ΔN: ΔN-ClpB*). …

Figure 6 with 3 supplements
Stabilizing ClpL ring dimers strongly reduces disaggregation activity.

(A) AlphaFold2 model of L. monocytogenes (Lm) ClpL ring dimers. Positions of individual domains are indicated. (B) Negative stain EM of Lm ClpL. Two-dimensional (2D) class averages revealing single …

Figure 6—source data 1

Source data includes the non-cropped and non-processed image of the SDS-gel.

https://cdn.elifesciences.org/articles/92746/elife-92746-fig6-data1-v2.zip
Figure 6—source data 2

Source data includes the non-cropped and non-processed image of the SDS-gel and indicates sections and loading schemata of the respective figure.

https://cdn.elifesciences.org/articles/92746/elife-92746-fig6-data2-v2.zip
Figure 6—figure supplement 1
ClpL rings interact in an M-domain-dependent manner.

(A) Oligomeric states of ClpL wild-type (WT) and indicated mutants were determined in the presence of ATP by size exclusion chromatography. ATPase-deficient ClpB-E279A/E678A (DWB) served as …

Figure 6—figure supplement 1—source data 1

Source data include non-cropped and non-processed images of SDS-gels.

https://cdn.elifesciences.org/articles/92746/elife-92746-fig6-figsupp1-data1-v2.zip
Figure 6—figure supplement 1—source data 2

Source data include non-cropped and non-processed images of SDS-gels and indicate sections and loading schemata of the respective figure supplement.

https://cdn.elifesciences.org/articles/92746/elife-92746-fig6-figsupp1-data2-v2.zip
Figure 6—figure supplement 2
Stabilizing ClpL ring dimers by disulfide crosslinking.

(A) Relative ATPase activities of indicated ClpL wild-type (WT) and T355C were determined at 0.125 and 1 μM protein concentration. The ATPase activities of ClpL-WT were set to 1. (B) Relative …

Figure 6—figure supplement 3
Production of ClpL M-domain (MD) mutants cause increased toxicity in E. coli.

(A) Production levels of ClpL (wild-type [WT] and indicated mutants) in E. coli ΔclpB cells. E. coli ΔclpB cells harboring plasmids for IPTG-controlled expression of clpL (WT and indicated mutants) …

Figure 6—figure supplement 3—source data 1

Source data includes the non-cropped and non-processed image of the SDS-gel.

https://cdn.elifesciences.org/articles/92746/elife-92746-fig6-figsupp3-data1-v2.zip
Figure 6—figure supplement 3—source data 2

Source data includes the non-cropped and non-processed image of the SDS-gel and indicates sections and loading schemata of the respective figure supplement.

https://cdn.elifesciences.org/articles/92746/elife-92746-fig6-figsupp3-data2-v2.zip
Author response image 1
Serial dilutions (10-1 – 10-6) of E.

coli dnaK103 mutant cells expressing E. coli dnaK, L. monocytogenes clpL or P. aeruginosa clpG were spotted on LB plates including the indicatedIPTG concentrations. Plates were incubated at 30°C or …

Author response image 2
LN-ClpB* cooperates with DnaK in protein disaggregation.

Relative MDH disaggregation activities of indicated disaggregation systems were determined. KJE: DnaK/DnaJ/GrpE. The disaggregation activity of Lm ClpL was set to 1. Statistical Analysis: Oneway …

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