Extended Data Fig. 4: Nucleotide binding and hydrolysis by PspA wild-type (WT) and mutants.
From: Structural plasticity of bacterial ESCRT-III protein PspA in higher-order assemblies
A: SDS PAGE of purified PspA WT and mutants (3 µg each). Purified proteins show a band at 28 kDa, except for the α0 truncated form which shows a band at 25 kDa. Asterisk: Faint dimer bands at twice the height of the monomeric protein band (56 kDa for WT and full-length PspA mutants; 50 kDa for α1-5 mutant). Marker: Bio-Rad Precision PlusProteinTM Unstained standards. B: ATPase activity of PspA. Left graph: Influence of Mg2+ and EDTA on the ATPase activity of the WT protein. WT (orange): ATPase activity in the presence of 2 mM Mg2+. -Mg2+(green): ATPase activity in the absence of Mg2+. EDTA (purple): ATPase activity in the presence of 10 mM EDTA; n(WT) = 36, n(-Mg2+ and EDTA) = 3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Middle graph: Comparison of the PspA ATPase activity with ATPase activity of E. coli PspF, n(PspA)=36, n(EcoPspF)=3. The boxplots show the mean ± standard error as boxes, the 10% to 90% percentile as whiskers, and outliers as diamonds. Right graph: Influence of ATPyS and AMPPCP on the ATPase activity of PspA. Red circle: ATPyS; Grey square: AMPPCP; n = 3, error bars represent SD. C: HPLC/MS-MS of PspA directly after purification. D: HPLC/MS-MS of PspA R44K/E126Q/E179Q + ATP after 24 h incubation and extensive washing. Different color lines represent different MRM transitions. For ADP, MRM transitions are 426/134 (red) and 426/159 (light red). For ATP MRM transitions are 506/79 (dark grey) and 506/159 (light grey). The ADP fragments below the ATP peak are formed by in-source fragmentation of ATP in the ESI source. E: Bar plot of estimated nucleotide concentrations found by LC-MS/MS in PspA apo control, WT, and R44K/E126Q/E179Q rods after incubation with ATP for 24 h and extensive washing, n = 1.
