Allosteric regulation of a prokaryotic small Ras-like GTPase contributes to cell polarity oscillations in bacterial motility
Fig 4
MglB Ct-helix facilitates nucleotide exchange of MglA.
(A) Kinetic measurements of increase in m-GDP fluorescence (phase I) upon addition of MxMglA (green), mix of MxMglA and MxMglB in 1:2 ratio (dark purple), mix of MxMglA and MxMglBΔCt in 1:2 ratio (magenta) at 400 seconds (marked by solid grey line labeled “+ P”), followed by competition of m-GDP (phase II) by addition of excess of unlabeled GDP at 1,800 seconds (marked by solid gray line labeled “+ GDP”). (B) Kinetic measurements of increase in m-GDP fluorescence (phase I) upon addition of MxMglA (green), mix of MxMglA and MxMglB in 1:2 ratio (dark purple), mix of MxMglA and MxMglBΔCt in 1:2 ratio (magenta) at 400 seconds (marked by solid gray line and labeled “+ P”), followed by competition of m-GDP (phase II) by addition of excess of unlabeled GTP at 1,800 seconds (marked by solid gray line labeled “+ GTP”). (C) Kinetic measurements of increase in m-GNP fluorescence (phase I) upon addition of MxMglA (green), mix of MxMglA and MxMglB in 1:2 ratio (purple), mix of MxMglA and MxMglBΔCt in 1:2 ratio (magenta) at 400 seconds (marked by solid grey line labeled “+ P”), followed by competition of m-GNP (phase II) by addition of excess of unlabeled GTP at 1,800 seconds (marked by solid gray line labeled “+ GTP”). ND for the kon and koff values in panel C stands for “not determined”. For panels A to C, phase I (between solid gray line labeled “+ P” and the first dotted line) represents kinetics for association (for kon estimation), phase II (between solid line labeled “+ GDP/GTP” and final dotted line) represents kinetics for dissociation (for koff estimation). A schematic representation of the observations and probable explanations at each phase is shown on the right side, following the same color scheme for the font as in the plots. The events directly observable through fluorescence intensity change of the mant-labeled component in the experiment are highlighted in bold. The panels include representative plots of multiple repeats, and the kon and koff values (in units of s-1) represent the average values of the multiple repeats. The numerical data for all the figure panels have been provided in the respective sheets in S1 Data. Ct-helix, C-terminal helix; m-GDP, 2’/3’-O-(N-Methyl-anthraniloyl)-guanosine-diphosphate; MxMglA, M. xanthus MglA; m-GNP, 2’/3’-O-(N-Methyl-anthraniloyl)-guanosine-5’-[(β,γ)-imido]triphosphate; MxMglB, M. xanthus MglB; MxMglBΔCt, M. xanthus MglB with Ct-helix truncated; ND, not determined.