Fig. 4: Cooperative ParB and DNA binding induces structural changes at the catalytic site of ParA.
From: Molecular basis of ParA ATPase activation by the CTPase ParB during bacterial chromosome segregation
a Hydrogen-deuterium exchange (HDX) analysis of the effect of ParB1-20 on wild-type ParA dimers. Shown is the difference in deuterium uptake by ParA (50 µM) in deuterated buffer containing ATP (1 mM) in the presence and absence of ParB1-20 (1 mM), mapped onto the crystal structure of the ParA21-274-D60A•ATP dimer in surface representation (t = 1000 s; Supplementary Fig. 9a and Supplementary Data 1). b Nucleotide content analysis investigating the effect of DNA and ParB1-20 on the ATPase activity of ParA under single-turnover conditions. Wild-type ParA or ParA-D60A (50 µM) was incubated with salmon sperm DNA (1 mg/mL) and/or ParB1-20 (1 mM) for 30 min at room temperature. After denaturation of the proteins, the released nucleotides were separated by HPLC and detected at a wavelength of 260 nm. The data show a representative experiment (n = 2). c HDX analysis investigating the structural changes in ParA-D60A dimers induced by ParB and DNA binding. ParA-D60A (50 µM) was incubated in deuterated buffer containing ATP and CTP (1 mM each) either alone or in the presence of salmon sperm DNA (1 mg/mL) and/or ParB-Q52A (ParB*) (100 µM) pre-incubated with a parS-containing DNA stem-loop (2.5 µM) to induce its transition to the closed state. The heatmap shows the maximal differences in deuterium uptake by ParA-D60A in the DNA-bound, ParB*-bound and DNA/ParB*-bound states compared to the apo-state for selected residues in the Walker A and Walker B regions, in a region at the dimer interface linking the DNA-binding site and the Walker A loop, and in the DNA-binding region (see Supplementary Fig. 9c and Supplementary Data 1 for details). (d) Global changes in the HDX pattern of ParA-D60A dimers upon incubation with DNA and/or ParB*. Shown are the maximal differences in deuterium uptake for each of the comparisons described in (c) mapped onto the crystal structure of the ParA21-274-D60A•ATP dimer in cartoon representation. ATP molecules are shown in orange, Mg2+ ions in green (see Supplementary Fig. 9c and Supplementary Data 1 for details). e Crystal structure of ParA21-274-D60A•ATP with a modeled ParB1-20 peptide taken from the predicted structure in Fig. 2f, shown in cartoon representation. ATP is depicted in orange, the Mg2+ ion in green. Hydrophobic residues in ParB1-20 (blue) and the Walker B-proximal loop of ParA (light red) are shown in stick representation. The Walker A (purple) and Walker B (magenta) motifs are highlighted, with catalytically relevant residues shown as sticks. Source data are provided as a Source data file.
