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. 2017 Nov 22;6:e30120. doi: 10.7554/eLife.30120

Figure 5. ClpC forms a large, inactive resting state that is sensitive to MecA and MD mutation.

(A) Glutaraldehyde crosslinking of ClpC-E280A/E618A (ClpC-DWB) and a respective MD mutant variant (F436A) was performed in absence and presence of ATP without and with MecA as indicated. Crosslinking of E. coli ClpB in presence of ATPγS served as reference defining crosslinked hexameric assemblies. Crosslink products were analyzed by SDS-PAGE. (B) Glutaraldehyde crosslinking of ΔN-ClpC and ΔN-ClpC-F436A was performed in absence and presence of ATPγS as indicated. Crosslinking of E. coli ClpB in presence of ATPγS served as reference defining crosslinked hexameric assemblies. Crosslink products were analyzed by SDS-PAGE. (C) Oligomeric states of ClpC-DWB and ClpC-F436A-DWB were determined in absence and presence of ATP. Addition of MecA and casein is indicated. Elution fractions were analyzed by SDS-PAGE and quantified. Positions of peak fractions of a protein standard and ClpB-E279A/E678A hexamers (+ATP) are indicated. (D) Micrograph of ClpC-F436A sample in presence of casein (top). Examples of single particles are circled. 2D class averages of ClpC-F436A (bottom). Scale bar is 10 nm. (E) Binding of FITC-casein to ClpC-DWB and ClpC-F436A-DWB was analyzed in presence of ATP by size exclusion chromatography. FITC-casein fluorescence of elution fractions was quantified. Positions of peak fractions of a protein standard and ClpB-E279A/E678A hexamers (+ATP) are indicated.

Figure 5.

Figure 5—figure supplement 1. MecA is crosslinked to a high molecular weight complex upon ClpC binding.

Figure 5—figure supplement 1.

Glutaraldehyde crosslinking of MecA, ClpC-E280A/E618A (ClpC-DWB) and both proteins together was performed in presence of ATP. MecA crosslink products were analyzed by SDS-PAGE and immunoblot analysis using MecA-specific antibodies.
Figure 5—figure supplement 2. ClpC-ATP forms an inactive large resting state.

Figure 5—figure supplement 2.

(A) Oligomeric states of ClpC-DWB and ClpC-F436A-DWB were determined in absence and presence of ATP by size exclusion chromatography (Superose6 HR10-30). Addition of MecA and casein is indicated. The elution profile of ATPase-deficient ClpB-E279A/E678A mutant (ClpB-DWB) served as reference for hexamer formation. Elution fractions were analyzed by SDS-PAGE. Positions of peak fractions of a protein standard are indicated. (B) ClpC-DWB and ClpC-F436A-DWB were subjected to size exclusion chromatography (Superose6 HR10-30). in presence of ATP. Addition of MecA and casein is indicated. Molar masses of complexes were determined by static light scattering measurements. Molecular weights (bold lines, left axis) and the scatter signal (thin lines, right axis) of complexes were plotted versus the elution volume. The determined molecular weight (Mw) of the peak fraction of the respective ClpC elution profiles and the derived ClpC oligomeric state (masses of MecA and casein were substracted) are given. (C) Complex formation between ClpP and ClpC-DWB or ClpC-F436A-DWB was analyzed by size-exclusion chromatography (Superose6 HR10-30). Addition of MecA is indicated. Elution fractions were analyzed by SDS-PAGE and quantified. (D) ClpC-F436A but not ClpC wild type efficiently binds substrate FITC-casein. Binding of ClpC wild type (WT) and ClpC-F436A to FITC-casein. Increasing concentrations of proteins were incubated with 0.1 μM FITC-casein in presence of 2 mM ATPγS and fluorescence polarization was recorded. Addition of MecA is indicated. (E) Binding of 5 μM ClpC wild type (WT) and ClpC-F436A to 0.1 μM FITC-casein in absence and presence of 2 mM ATPγS. Changes in fluorescence polarization were recorded.
Figure 5—figure supplement 3. Autodegradation of ClpC MD mutants.

Figure 5—figure supplement 3.

ClpC wild type (WT) and indicated MD mutants were incubated with ClpP and an ATP regenerating system. Samples were analyzed after ATP addition at the indicated time points by SDS-PAGE. A protein standard is given.