Fig 1.
Localization of cell polarity determining proteins in M. xanthus.
Schematic showing the localization of MglA, MglB, and RomRX in M. xanthus. MglA (GTP-bound state) localizes to the leading pole, whereas MglB and RomRX mainly localize at the lagging pole. A polarity reversal occurs when the proteins relocalize. A signaling cascade consisting of the Frz-signaling proteins drives the polarity reversals. MglA, MglB, and RomRX are represented as green, magenta, and orange spheres. The asymmetry in the size of orange spheres at the 2 poles denotes the asymmetric localization of RomR, with higher concentration at the lagging pole. FrzXP and FrzZP denote phosphorylated states of FrzX and FrzZ, respectively. Frz, frizzy; Mgl, mutual gliding motility.
Fig 2.
Crystal structures of M. xanthus MglA and MglAB–GTPγS complex.
(A) Crystal structure of MxMglA bound to GDP. The secondary structure elements are labeled. GDP is shown in stick representation in light brown, and the catalytic loops switch 1 (blue) and switch 2 (red) are labeled as SW1 and SW2. The rest of the loops are shown in light pink, whereas the secondary structure elements are shown in shades of green. The C-terminal hexa-histidine tag (His)6, which is ordered in the crystal structure, is shown in gray. (B) Crystal structure of the complex of MxMglA (green) bound to GTPγS (yellow) and MxMglB (magenta). “Front” view and a 90° rotated view (side view) are shown. The 2 protomers of MxMglB are labeled as MglB1 and MglB2. The C-terminal helix of MxMglB (Ct-helix), the respective N- and C-terminal ends, switch 1 (blue; SW1) and switch 2 (red; SW2), and relevant secondary structures of MxMglA are labeled. GTPγS is shown in stick representation in yellow. Dotted magenta line connects the ends on either sides of the stretch of disordered residues in MglB1. Boxed regions in the front and side view panels highlight the major interfaces between MxMglA with the Rbl/LC7 domain of MxMglB (box with short dashes) and the Ct-helix of MxMglB (box with long dashes), respectively. (C) Residues of β2 strand (Phe56A, Phe57A, and Phe59A) of MxMglA (green) form a hydrophobic surface that interacts with MxMglB (magenta). The relevant secondary structure elements are labeled, and switch 1 and 2 (SW1 and SW2) are colored in blue and red, respectively. The region corresponds to a zoomed view of the boxed region (short dashes) in the front view of panel B. (D) The interacting interface of the Ct-helix of MxMglB (magenta) and MxMglA (green). The side chains of relevant interface residues are shown in stick representation and labeled. Water molecules are represented as red spheres, and dotted yellow lines represent hydrogen bond interactions. The region corresponds to a zoomed view of the boxed region (long dashes) in the side view of panel B. (E and F) Conformational changes in switch 1 (SW1; 2 shades of blue) and switch 2 (SW2; 2 shades of red) in the presence of MxMglB illustrates the mechanism of GAP activity (by reorientation of active site residues Arg53A and Glu82A). MxMglA–GDP conformation and MglA from the MxMglAB–GTPγS complex are shown in panels E and F, respectively. The 2 structures from a superposed orientation are shown as separate panels. Secondary structures extending from the switch loops for MxMglA–GDP conformation and MglA from the MxMglAB–GTPγS complex are shown in brown and green, respectively. Switch 1 (or 2) of the 2 structures are labeled SW1 (or 2) and SW1 (or 2) using GDP and GSP as subscripts for the MxMglA–GDP and MxMglAB–GTPγS complexes, respectively. (G and H) Ct-helix of MxMglB (magenta) and GTP (represented by GTPγS shown in stick representation) bind to the C- and N-terminal ends of the β2 strand of MxMglA, respectively. MxMglA–GDP conformation and MglA from the MxMglAB–GTPγS complex are shown in panels G and H, respectively. The 2 structures from a superposed orientation are shown as separate panels. The unflipped and flipped states of the strand are shown (brown, MxMglA–GDP; green, MglA conformation in MxMglAB–GTPγS structure; switch 1 and switch 2 are in shades of blue and red, respectively). A schematic representation of the registry shift and β2 flip is shown below, with the same color scheme. A 6-pointed yellow star and a 5-pointed light brown star represent GTP (labeled as “T’) and GDP (labeled as “D”), respectively. The β2 strand is represented by an arrow. The residues that form the interface are schematically shown by 2 circles on the bottom or top of the β2 strand representation in the flipped and unflipped states of the strand, respectively. Ct-helix of MxMglB is schematically shown by a cylinder that is labeled “H.” Ct-helix, C-terminal helix; GAP, GTPase activating protein; GTPγS, guanosine 5’-O-[gamma-thio]triphosphate; Mgl, mutual gliding motility; MxMglA, M. xanthus MglA; MxMglB, M. xanthus MglB; Rbl, Roadblock; SW1, switch 1; SW2, switch 2.
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.
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.
Fig 5.
MxMglB Ct-helix is required for MxMglB function in vivo.
(A) Expression of MxMglBΔCt leads to motility defects. Motility on 0.5% agar is shown after 48 hours of incubation at 32°C. Scale bar = 1 cm. (B) Reversal frequency of M. xanthus cells of ΔmglB mutant strain and cells expressing wild-type MxMglB (mglB+) or MxMglBΔCt (mglBΔCt+). The reversal frequencies were scored in absence (−) or presence (+) of 0.075% IAA, a condition known to activate the Frz system. For each strain and condition, the number of trajectories analyzed (n), the median (vertical line), and the mean (black dot) are reported for each box-and-whisker plot. (C) MxMglBΔCt shows a bipolar localization pattern. Representative fields of cells expressing MxMglBΔCt-nG and MxMglB-nG are shown in top and bottom images, respectively. The fractions of cells exhibiting bipolar (dark blue), unipolar (light blue), and diffuse (cyan) localization are depicted on the left for each of the field views, in which “n” represents the total number of cells analyzed. The fluorescence intensity profiles for each cell are compiled and shown in S4A Fig. (D) MxMglA shows a bipolar localization pattern in the presence of MxMglBΔCt. Representative fields of cells expressing MxMglA-YFP in mglBΔCt+ and mglB+ strains are shown in top and bottom images, respectively. The fractions of cells exhibiting bipolar, unipolar, and diffuse localization are depicted on the left for each of the field views, in which “n” represents the total number of cells analyzed. The fluorescence intensity profiles for each cell are compiled and shown in S4B Fig. (E) RomR shows a bipolar localization pattern in the presence of MxMglBΔCt. Representative fields of cells expressing RomR-GFP in mglBΔCt+ and mglB+ strains are shown in top and bottom images, respectively. The fractions of cells exhibiting bipolar, unipolar, and diffuse localization are depicted on the left for each of the field views, in which “n” represents the total number of cells analyzed. In the bottom panel (RomR-GFP, mglB+), dashed lines indicate bacterial contour. The fluorescence intensity profiles for each cell are compiled and shown in S4B Fig. The numerical data for all the figure panels have been provided in the respective sheets in S1 Data. Ct-helix, C-terminal helix; Frz, frizzy; IAA, isoamyl alcohol; MxMglA, M. xanthus MglA; MxMglB, M. xanthus MglB; MxMglBΔCt, MxMglB with Ct-helix truncated; nG, neonGreen; YPF, yellow fluorescent protein.
Fig 6.
Conservation of sequence features of the allosteric pocket on MglA and MglB Ct-helix.
(A) Comparison of residue conservation in C-terminal extension of coupled versus orphan MglB sequences. The aspartate residues implicated in interaction with the allosteric pocket on MglA are highlighted by “*” in the sequence conservation logo. The fractional occupancy of amino acids in the sequence alignment is shown for each amino acid position. The numbers within brackets denote the number of sequences in the alignment. The residues are colored in 20 different shades. (B) Comparison of residue conservation in the α5 helix of MglA sequences coupled with MglB sequences possessing a negatively charged C-terminal extension versus that of all coupled MglA sequences. The lysine residues that potentially interact with the Ct-helix of MglB are highlighted by “*” in the sequence conservation logo. The fractional occupancy of amino acids in the sequence alignment is shown for each amino acid position. The numbers within brackets denote the number of sequences in the alignment. Ct-helix, C-terminal helix; MglA, Mutual Gliding motility A; MglB, Mutual Gliding motility B.
Fig 7.
Ct-helix regulates MxMglAB interaction by binding to an allosteric pocket of the small Ras-like GTPase fold.
(A) A schematic representation of the conditions that facilitate MxMglAB complex formation. PDB IDs 6IZW, 3T12, and 5YMX correspond to the crystal structures of MxMglAB–GTPγS, TtMglAB–GTPγS, and MxMglA–GDP complexes. (B) Proposed model for MxMglAB relocalization. Proteins that can bind in the allosteric pocket potentially compete out the MxMglB Ct-helix and dissociate the MxMglAB–GDP complex. In panels A and B, MxMglB dimer is shown in magenta, whereas MxMglA conformations with the flipped and unflipped states of β2 are shown in green and brown, respectively. A 6-pointed yellow star and a 5-pointed light brown star represent GTP (labeled as “T”) and GDP (labeled as “D”), respectively. The β2 strand is represented by an arrow. The residues that form the interface are schematically shown by 2 circles on bottom or top of the β2 strand representation in the flipped and unflipped states of the strand, respectively. Ct-helix of MxMglB is schematically shown by a cylinder and labeled as “H.” Ct-helix, C-terminal helix; MxMglA–GDP, MxMglA bound to guanosine 5’-diphosphate; MxMglA, M. xanthus MglA; MxMglAB, M. xanthus MglA and MglB complex; MglAB–GTPγS, MglA and MglB complex formed in the presence of guanosine 5’-O-[gamma-thio]triphosphate; PDB ID, Protein Data Bank identification.