Fig. 5: Residue R13 of ParB is dispensable for ParA binding but critical for ATPase stimulation.
From: Molecular basis of ParA ATPase activation by the CTPase ParB during bacterial chromosome segregation
a BLI analysis of the interaction of ParA with DNA-bound ParB-Q52A (ParB*) variants lacking the conserved residue R13. The indicated ParB-Q52A variants were loaded onto a closed parS-containing DNA fragment (see Fig. 1a) and probed with ParA-R238E (ParA*) (5 µM) in the presence of ATP and CTP (1 mM each). At the end of the association phase, the biosensors were transferred into nucleotide- and protein-free buffer to monitor the dissociation reactions. Shown are the results of a representative experiment (n = 3 independent replicates). b Stimulation of the ParA ATPase activity by ParB-Q52A (ParB*) variants lacking R13. Shown is the ATPase activity of ParA (5 μM) in the presence of increasing concentrations of the indicated ParB-Q52A variants in reactions containing ATP (1 mM), CTP (1 mM), salmon sperm DNA (100 µg/mL) and a parS-containing DNA stem-loop (250 nM). Data represent the mean of four independent replicates (±SD), normalized to reactions without ParB*. The results were fitted to a Hill equation. c Subcellular localization of ParB variants lacking R13 in M. xanthus. Cells producing Tq-ParB (MO072), or Tq-ParB-R13K (LS005) or Tq-ParB-R13A (LS004) in place of wild-type ParB were stained with DAPI prior to analysis by phase contrast and fluorescence microscopy. The images show overlays of the Tq and DAPI signals, with the cell outlines indicated in white (bar: 5 µm). The demographs on the right summarize the subcellular distribution of the Tq signal in representative subpopulations of cells (n = 400 per strain). d Quantification of the number of fluorescent foci in cells producing wild-type (WT) Tq-ParB (MO072, n = 358) or its R13K (LS005, n = 422) or R13A (LS004, n = 369) derivative in place of the native ParB protein. e Immunoblot analysis of the strains analyzed in panel c with anti-GFP antibodies. A ΔparB mutant producing untagged ParB under the control of an inducible promoter (SA4269) was used as a negative control (−). Shown is a representative image (n = 3 independent replicates). Source data are provided as a Source data file.
