Allosteric regulation of a prokaryotic small Ras-like GTPase contributes to cell polarity oscillations in bacterial motility
Fig 3
Ct-helix of MxMglB allosterically regulates GTPase activity of MxMglA.
(A) GTPase activities of wild-type MxMglA only (green), and in the presence of MxMglB (dark purple; MglAB), and MxMglBΔCt (magenta; MglABΔCt). Ratio variations of 1:2 and 1:10 for MxMglBΔCt are shown in dotted and solid lines, respectively. The release of GDP was estimated using NADH-based enzyme-coupled assay. The lines represent the average of multiple repeats (at least 3), and the shaded zones represent standard error. (B) Comparison of kcat values for MxMglA, MxMglAB, MxMglABΔCt, MxMglAB3D, and MxMglAQB. The release of GDP was estimated using NADH-based enzyme-coupled assay. 1:2 and 1:10 represent the molar ratio of MxMglA and MxMglB monomers. (C) Comparison of kcat values for MxMglA, MxMglAB, MxMglABΔH6 (MglB construct without a hexahistidine tag) and MxMglABΔCt. The release of phosphate was estimated using malachite green assay. 1:2 represents the molar ratio of MxMglA and MxMglB/MxMglBΔH6/MxMglABΔCt monomers. (D) Comparison of kcat values for MxMglA, MxMglAK, and MxMglAL in the presence of MxMglB. The release of GDP was estimated using NADH-based enzyme-coupled assay. 1:2 and 1:10 represent the molar ratios of MxMglA and MxMglB monomers. The data shown for MxMglA and MxMglAB have been duplicated from panel B for the sake of comparison. (E) Fluorescence anisotropy measurements for MxMglB (dark purple) and MxMglBΔCt (magenta) titrated against MxMglA–m-GNP, showing that both MxMglB and MxMglBΔCt bound to MxMglA in the presence of m-GNP. (F) Fluorescence anisotropy measurements for MxMglB (dark purple) and MxMglBΔCt (magenta) titrated against MxMglA–m-GDP, showing that MxMglB, but not MxMglBΔCt, bound to MxMglA in the presence of m-GDP. The mean and 95% confidence intervals (long and short black horizontal lines, respectively) are shown for each sample in panels B, C and D. *p = 0.01–0.05, **p = 0.001–0.01, and ***p < 0.001. The numerical data for all the figure panels have been provided in the respective sheets in S1 Data. Ct-helix, C-terminal helix; m-GNP, 2’/3’-O-(N-Methyl-anthraniloyl)-guanosine-5’-[(β,γ)-imido]triphosphate; MxMglABΔH6, Mx MglA and MglB (without hexahistidine tag) complex; MxMglAB3D, Mx MglA and MglB D150A, D151A and D152A triple mutant); MxMglAQB, MxMglA Q82L mutant; MxMglA, M. xanthus MglA; MxMglAK, MxMglA K181A and K185A double mutant; MxMglAL, MxMglA L64A and I67A mutant; MxMglB, M. xanthus MglB; MxMglBΔCt, MxMglB Ct-helix truncation; ns, non-significant.