Fig. 2: VirB is a CTP-binding protein.
a, b Isothermal titration calorimetry analysis of the interaction of VirB with a CTPγS and b CDP. A solution of VirB (115 µM) was titrated with a stock solution (1.55 mM) of the indicated nucleotides. The graphs indicate the heat changes observed after each of the 13 injections. The KD values obtained are given in the graphs. c Microscale thermophoresis analysis of the interaction of VirB (WT) and its R93A and R94A variants with CTP, CDP and ATP. Bars show the mean equilibrium association constants (1/KD) obtained for the indicated conditions (±SD, when indicated). Data points (diamonds) represent the results of n = 3 (CTP binding to WT and R93A) or 2 (all other reactions) independent experiments, each of which was performed in triplicate. The underlying titration curves are given in Supplementary Fig. 3c. The corresponding KD values are: WT-CTP (11 µM), WT-CDP (88 µM), and R93A-CTP (1.3 mM). n.d. not detectable. d CTPase activities of VirB and MxParB. The indicated proteins (5 µM) were incubated with 1 mM CTP and/or double-stranded DNA oligonucleotides containing a virS site (0.5 µM), a scrambled virS site (0.5 µM) or an M. xanthus parS site (0.3 µM). CTP hydrolysis rates were determined using an NADH-coupled enzyme assay. The values obtained were corrected for the background activity measured in reactions containing only the respective protein but no ligands. Columns represent the mean (±SD) of three independent experiments (diamonds), each of which was performed in triplicate. The statistical significance of differences between reactions was determined using an unpaired two-sided Welch’s t-test. Relevant p values are given in the graph. Source data are provided as a Source Data file.
