Fig. 1: ParA binds the N-terminal region of ParB with a preference for closed ParB clamps.

a Schematic of the biolayer interferometry (BLI) setup used to study the interaction of ParA with ParB. Double-biotinylated DNA fragments (234 bp) containing a central M. xanthus parS site were immobilized on a streptavidin-coated biosensor. Subsequently, ParB or its CTP hydrolysis-deficient ParB-Q52A variant (10 µM) was loaded onto the closed fragments in the presence of CTPγS or CTP (1 mM), respectively, to form stable model partition complexes, which were then analyzed for their interaction with ParA. b BLI analysis of the interaction of ParA with DNA-bound ParB. Wild-type ParB was loaded onto closed parS-containing DNA as described in panel a and probed with the indicated concentrations of ParA-R238E (ParA*) in the presence of ATP (1 mM). At the end of the association phase, the biosensor was transferred into protein- and nucleotide-free buffer (Wash) to follow the dissociation kinetics. Shown is a representative experiment (n = 3). c Conservation of the N-terminal region of ParB. Shown are a schematic depicting the domain organization of ParB and a sequence logo showing the conserved ParA-binding motif, based on an alignment of 3800 ParB homologs obtained by protein BLAST analysis with M. xanthus ParB as a query. Residues are colored according to their physico-chemical properties (black: hydrophobic, blue: positively charged, red: negatively charged, green: polar). d BLI analysis investigating the role of the N-terminal region of ParB in the ParA-ParB interaction. ParB or ParBΔ21 were loaded onto closed parS-containing DNA as described in (a) and probed with ParA-R238E (ParA*) (10 µM) in the presence of ATP and CTP (1 mM each). Shown is a representative experiment (n = 3 independent replicates). e Subcellular localization of ParB and ParBΔ21 in M. xanthus. Cells producing Tq-ParB (MO072) or Tq-ParBΔ21 (LS007) in place of wild-type ParB were stained with 4′,6-diamidino-2-phenylindole (DAPI) prior to analysis by phase contrast and fluorescence microscopy. The images show overlays of the sfmTurquoise2^ox (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). The single-cell fluorescence profiles were sorted according to cell length and stacked on top of each other. f Role of the opening state of ParB clamps in the stimulation of the ParA ATPase activity. The graphs show that ATPase activities of ParA (5 μM) incubated with ParB-Q52A (ParB*) (10 µM) in a buffer containing ATP (1 mM) and salmon sperm DNA (100 µg/mL) in the absence (“open”) or presence (“closed”) of CTP (1 mM) and a short parS-containing DNA stem-loop (250 nM). A reaction containing closed ParBΔ21 clamps served as a negative control (Δ21). Data represent the mean of three independent replicates (±SD). The results were fitted to a Hill equation to account for the sigmoidal shape of the binding curve. However, note that for the first data points, [ParB*] is within the range of the ParA concentration and close to the K_D of the ParA-ParB interaction (see b), leading to titration of the free ligand species. Moreover, due to the close linkage of the N-terminal peptides in the ParB dimer, the reactions may be influenced by avidity effects. Therefore, it is not straightforward to determine whether the binding behavior observed actually signifies cooperativity in the binding process. Source data are provided as a Source data file.