Structural basis of flagellar motility regulation by the MogR repressor and the GmaR antirepressor in Listeria monocytogenes

Abstract

The pathogenic Listeria monocytogenes bacterium produces the flagellum as a locomotive organelle at or below 30°C outside the host, but it halts flagellar expression at 37°C inside the human host to evade the flagellum-induced immune response. Listeria monocytogenes GmaR is a thermosensor protein that coordinates flagellar expression by binding the master transcriptional repressor of flagellar genes (MogR) in a temperature-responsive manner. To understand the regulatory mechanism whereby GmaR exerts the antirepression activity on flagellar expression, we performed structural and mutational analyses of the GmaR-MogR system. At or below 30°C, GmaR exists as a functional monomer and forms a circularly enclosed multidomain structure via an interdomain interaction. GmaR in this conformation recognizes MogR using the C-terminal antirepressor domain in a unique dual binding mode and mediates the antirepressor function through direct competition and spatial restraint mechanisms. Surprisingly, at 37°C, GmaR rapidly forms autologous aggregates that are deficient in MogR neutralization capabilities.

Figure 1.

Intermolecular interaction between the GmaR antirepressor and the transcriptional repressor MogR, and the inhibitory effect of GmaR on the MogR–dsDNA interaction. (A) Schematic representation of the MogR proteins (MogR_FL, MogR_D, MogR_1-151 and MogR_1-135) used in this study. The antirepressive and repressive sites of MogR are indicated below the schematic diagram of MogR_FL that defines the MogR domains. A black triangle on MogR_D represents the insertional mutation site used to generate the MogR_D^49ins, MogR_D^34ins and MogR_D^19ins mutants (Figure 7B). (B) Schematic representation of the GmaR proteins (GmaR_FL, GmaR_141-637 and GmaR_231-637) used in this study. The antirepressive sites and interdomain interaction sites of GmaR are indicated below the schematic diagram of GmaR_FL that defines the GmaR domains. Black dots in GmaR_FL and GmaR_141-637 represent the GmaR residues (E293, H286 and R589) that were mutated to confirm the contribution of the interdomain interaction to protein thermostability. (C–E) GmaR–MogR complex formation. The direct interaction of GmaR_FL with MogR_D was analyzed by gel-filtration chromatography (C), native PAGE (D) and ITC (E; Wiseman c-value, 2068 ± 684) (42). The data are representative of three independent experiments that yielded similar results. (F) GmaR-mediated inhibition of the MogR_D–dsDNA interaction in EMSA (n = 3 independent experiments). The molar ratio in each reaction is shown above the gel image.

Figure 2.

GmaR^Apo structure and its interdomain contacts. (A) Crystal structure of GmaR^Apo containing the TPR, linker and antirepressor domains. The GmaR^Apo structure is shown as rainbow ribbons [N-terminus (N-term), blue; C-terminus (C-term), red]. The α-helices of GmaR^Apo are numbered in domain-specific colors (TPR, blue; linker, yellow; antirepressor, red). The GmaR^Apo structure is also depicted in a box as rainbow ribbons with transparent surfaces in a 55°-rotation view of the left figure to highlight the circularly enclosed conformation of GmaR. (B) Interdomain contacts of GmaR^Apo between the TPR and antirepressor domains. Interdomain interface residues in the TPR (cyan ribbons) and antirepressor (magenta ribbons) domains are shown as light blue and orange sticks, respectively, with transparent surfaces. Interdomain hydrogen bonds are represented by dashed lines.

Figure 3.

Crystal structure of the GmaR-MogR complex. (A) Overall structure of the GmaR–MogR complex. GmaR is depicted as a surface representation in domain-specific colors (TPR, cyan; linker, yellow; antirepressor, magenta). MogR is shown as green ribbons, and its disordered region (residues 137–151) is represented by green dotted lines. Two distinct antirepressive sites (sites 1 and 2) that correspond to the intermolecular interfaces between GmaR and MogR are highlighted by black dashed circles. The orientation of the figure is identical to that of the inset of Figure 2A. (B) Structural similarity of GmaR irrespective of MogR binding. The GmaR^MogR structure (magenta ribbons) is overlaid on the GmaR^Apo structure (light blue ribbons). (C) Structural comparison of MogR^GmaR with MogR^dsDNA. The main body of MogR exhibits similar structures irrespective of binding partners. However, the C-terminal extension of MogR adopts different structures depending on binding partners. The MogR^GmaR structure (green ribbons) is overlaid on the MogR^dsDNA structure (orange ribbons; PDB ID: 3FDQ).

Figure 4.

Intermolecular binding sites of GmaR and MogR. (A) Intermolecular interactions in the GmaR–MogR structure. GmaR is shown as ribbons in domain-specific colors (TPR, cyan; linker, yellow; antirepressor, pink). MogR is represented by light green ribbons. The intermolecular interface residues of GmaR and MogR are shown as magenta and green lines, respectively. Antirepressive sites 1 and 2 are highlighted by black dashed circles. The orientation of the figure is identical to that of Figure 3A. (B) Antirepressive site 1. The MogR residues at site 1 are depicted as green sticks in the C-terminal extension of MogR (light green coil). The GmaR residues at site 1 are shown as magenta sticks on the GmaR^MogR structure (pink ribbons and gray surfaces). Intermolecular hydrogen bonds are represented by black dashed lines. (C) Contributions of site-1 residues to GmaR–MogR binding based on the mutational analysis. MogR residues at site 1 (K152, T153, N154, I155 and F156) were individually mutated to alanine in MogR_D, and the GmaR_FL-binding capacities of the MogR_D mutants were analyzed by ELISA. The FliD protein was used as a negative control. The data are representative of three independent experiments that yielded similar results, and the EC₅₀ values represent the means ± S.D. from the three independent experiments. (D) Antirepressive site 2. The MogR and GmaR residues at site 2 are depicted as green and magenta sticks in the MogR (light green coils) and GmaR (pink ribbons and gray surfaces) structures, respectively. Intermolecular hydrogen bonds are represented by dashed lines. (E) Contributions of site-2 residues to GmaR–MogR binding based on the mutational analysis. ELISA was employed to determine the GmaR_FL-binding capacities of the site-2 mutants of MogR_D (MogR_D^T112A, MogR_D^S114A, MogR_D^Q117A, MogR_D^N118A, MogR_D^K122A and MogR_D^{S114A/Q117A/N118A/K122A}). The data are representative of three independent experiments that yielded similar results, and the EC₅₀ values represent the means ± S.D. from the three independent experiments.

Figure 5.

Primary contributions of spatially distinct antirepressive site 1 and repressive site MIG to the GmaR–MogR and MogR–dsDNA interactions, respectively. (A) Antirepressive GmaR-binding sites of MogR in the GmaR–MogR complex structure. MogR residues at antirepressive sites 1 and 2 are colored red and blue, respectively, on MogR (green cartoon) in complex with GmaR (gray cartoon). (B) Repressive dsDNA-binding sites of MogR in the MogR–dsDNA complex structure (PDB ID: 3FDQ). MogR residues at repressive sites MIG and MAG are colored orange and blue, respectively, on MogR (green cartoon) in complex with dsDNA (gray cartoon). (C) Schematic diagram of antirepressive and repressive sites in MogR_D. Antirepressive site 2 overlaps with repressive site MAG in the main body of MogR. However, antirepressive site 1 and repressive site MIG are segregated in the C-terminally extended coil structure of MogR. Three C-terminally truncated MogR proteins (MogR_D, MogR_1-151 and MogR_1-135) were used in binding assays to define the contribution of each site to GmaR or dsDNA binding. (D,E) Native PAGE (D) and ELISA (E) of GmaR_FL in the presence of the MogR_D, MogR_1-151 or MogR_1-135 variants to assess the contributions of antirepressive sites 1 and 2 to GmaR–MogR binding (n = 3 independent experiments). (F,G) EMSA (F) and FP assay (G) of the operator dsDNA in the presence of the MogR_D, MogR_1-151 or MogR_1-135 variants to define the contributions of repressive sites MIG and MAG to MogR–dsDNA binding (n = 4 independent experiments). (H,I) Critical role of antirepressive site 1 in the inhibitory activity of GmaR on the MogR–dsDNA interaction. The interaction of MogR_D or MogR_D^Site1-Ala with dsDNA was analyzed by EMSA (H; n = 3 independent experiments) and FP assay (I; n = 9 independent experiments) in the absence or presence of GmaR_FL.

Figure 6.

Direct competition mechanism used by antirepressive site 2 of GmaR to interfere with the MogR–dsDNA interaction. (A) MogR residues shared by antirepressive site 2 and repressive site MAG. The GmaR-binding residues of MogR at site 2, in particular from the H6 and H7 helices, are also used to interact with dsDNA as site MAG, indicating that GmaR directly competes with dsDNA to interact with site 2 of MogR. In the overlaid MogR^GmaR (light green ribbons) and MogR^dsDNA (light orange ribbons) structures, the MogR residues that are commonly located at sites 2 (green) and MAG (orange) are represented by sticks, and those observed at only one site are depicted as lines. (B) Negative electrostatic potentials of GmaR around the H7 helix of MogR at antirepressive site 2 in the GmaR–MogR complex structure. The electrostatic potentials of GmaR are shown as differently colored surfaces (negative, red surface; neutral, white surface; positive, blue surface). The H6 and H7 helices and their connecting loop of MogR are shown as light green ribbons.

Figure 7.

Spatial restraint mechanism used by antirepressive site 1 of GmaR to inhibit the MogR–dsDNA interaction. (A) Occlusion of MogR site MIG from dsDNA by the site 1 interaction. The GmaR–MogR and MogR–dsDNA structures are overlaid by superimposing the MogR structures as a reference point. Sites 2/MAG, site MIG and site 1 are colored blue, orange, and red, respectively, in the MogR structures (green) in complex with GmaR (magenta) or dsDNA (gray). In the overlaid structures, GmaR atoms between sites 1 and 2 make substantial steric clashes (black spheres) with dsDNA atoms, particularly at site MIG, suggesting that the site 1 GmaR–MogR interaction occludes MogR site MIG from binding dsDNA despite the distinct positions of sites 1 and MIG and inhibits the minor groove interaction through a spatial restraint mechanism. (B) Verification of the spatial restraint mechanism. To alleviate the spatial restraint caused by the site 1 interaction, 49, 34 or 19 additional residues were inserted between sites MIG and 1 in MogR_D to generate the MogR_D^49ins, MogR_D^34ins and MogR_D^19ins mutants, respectively. The interaction of MogR_D or its insertional mutants with dsDNA was analyzed by EMSA in the absence or presence of GmaR_FL (n = 3 independent experiments). The insertional mutations attenuated the inhibitory effect of GmaR_FL on the MogR_D–dsDNA interaction. Complex formation by MogR_D (left) or MogR_D^49ins (right) is schematically shown above the gel image. When MogR_D forms a heterodimer with GmaR, MogR_D does not interact with dsDNA due to the spatial restraint induced by the site 1 interaction. However, the MogR_D^49ins mutant simultaneously binds GmaR and dsDNA because of relieved spatial restraint.

Figure 8.

The 37°C-labile stability and function of GmaR. (A) 37°C-induced aggregation of GmaR_FL in native PAGE (n = 3 independent experiments). The monomeric GmaR_FL protein was incubated at 23, 30 or 37°C for various times and then analyzed by native PAGE to address a temperature-dependent change in the oligomeric state of GmaR_FL. (B) 37°C-induced aggregation of GmaR_FL in gel-filtration chromatography (n = 3 independent experiments). The monomeric GmaR_FL protein was incubated at 30 or 37°C for 5 or 15 min and then analyzed by gel-filtration chromatography to address a temperature-dependent change in the oligomeric state of GmaR_FL. (C) MogR-binding activity of GmaR at different temperatures. The GmaR_FL protein was incubated at 4, 30 or 37°C for 15 min, and then the interaction of the resulting GmaR_FL protein with MogR_D was analyzed by native PAGE (n = 4 independent experiments). (D) Inhibitory effect of GmaR on the MogR–dsDNA interaction depending on exposure temperature. The GmaR_FL protein was incubated at 4, 30 or 37°C for 15 min, and then the inhibition of the MogR–dsDNA interaction by the resulting GmaR_FL protein was analyzed by EMSA (n = 4 independent experiments). (E) Aggravated aggregation of GmaR at 30 and 37°C resulting from a loss of interdomain contacts at the GmaR E293 residue. The GmaR_FL and GmaR_FL^E293A proteins were incubated at 30 or 37°C, and their oligomeric states were analyzed by native PAGE (n = 3 independent experiments). (F) Alleviated aggregation of GmaR at 37°C by stabilizing the interdomain interaction between the GmaR H286 and R589 residues. The GmaR_141-637 and GmaR_141-637^H286C/R589C proteins were incubated at 30 or 37°C, and their aggregation profiles were determined by native PAGE (n = 3 independent experiments). GmaR_141-637 was used for the H286C and R589C mutations instead of GmaR_FL to avoid unexpected disulfide bond formation caused by three cysteine residues (residues 8, 87 and 139) in the glycosyltransferase domain. GmaR_141-637 exhibited thermostability similar to that of GmaR_FL.

Figure 9.

Proposed molecular model for the temperature-dependent regulation of flagellar expression by the GmaR–MogR system. (A) Flagellar expression through GmaR-mediated neutralization of MogR at or below 30°C outside the host. (B) GmaR aggregation and MogR-mediated suppression of flagellar expression at 37°C inside the host.