Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducingpeptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci

Mechanism of ComR activation by the XIP peptide.

<p>The empty XIP-binding pocket of the apo form of ComR is represented by an open triangle. Helix α9, which undergoes a major conformational change, is shown as a yellow stick. Key ComR residues are labeled, in black for XIP binding and activation residues (T90, Y91, R92, and K100), in red for residues involved in DNA binding and HTH sequestration (R35, R39, R51, E117, D146, and E147) and in blue for residues of the dimerization interface (K87, D200, K246, and E282).</p

Binding on ComR of WT and variants XIPs as measured by ITC.

<p>Binding on ComR of WT and variants XIPs as measured by ITC.</p

<i>In vivo</i> transcriptional activity.

<p><b>A–<i>In vivo</i> activities of ComR mutants affected in dimerization, XIP binding and activation mechanism.</b> Maximum specific luciferase activity (RLU OD₆₀₀^-1) emitted by the ComS^- reporter strain producing ComR WT (black bar) and ComR mutants K87A, D246A, K87A/D246A, T90A, Y91A, R92A, K100A, F171A/Y174A (gray bars) grown in presence of 50 nM XIP. <b>B–<i>In vivo</i> activities of ComR mutants affected in HTH sequestration.</b> Maximum specific luciferase activity of the ComS^- reporter strain producing ComR WT (black bars) and ComR mutants E117A/E118A and E146A/D147A grown without (white bars) or with 50 nM XIP (gray bars). Bars represent the average of three independent repeats ± standard deviation.</p

Thermodynamic signatures of the binding on ComR of XIP WT and variants.

<p>The enthalpy change (ΔH), the entropic contribution (-TΔS) and the Gibbs free energy change (ΔG) are shown in blue, green and red, respectively, and expressed in kcal mol^-1.</p

Apo conformation of ComR.

<p><b>A–Overall view of the monomeric apo state.</b> ComR is shown as cartoon colored by spectrum from TPR-1 in cyan to TPR-5 in red with the additional CAP helix α16 in purple and the HTH-domain in blue. The α-helices are labeled. <b>B–Close view of the HTH sequestration mode.</b> The protein is shown as cartoon colored by spectrum as in panel A with the contact residues highlighted in sticks, colored by atom type and labeled. <b>C–Superimposition of the free and bound forms of the TPR domain.</b> Superimposition of the apo structure of ComR, colored by spectrum, and chain B from the ComR/XIP/DNA dimer, shown in gray.</p

Thermodynamic signatures of XIP binding on ComR WT and mutant proteins.

<p>The enthalpy change (ΔH), the entropic contribution (-TΔS) and the Gibbs free energy change (ΔG) are shown in blue, green and red, respectively, and expressed in kcal mol^-1.</p

Sequence alignment of representative ComR orthologues.

<p>The ComR apo secondary structure elements of apo ComR are indicated at the top of the alignment. The α-helices are coloured according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005980#ppat.1005980.g001" target="_blank">Fig 1A</a>. Strictly conserved residues are highlighted with red background and white labels. Conserved residues are written in red in blue frames. The similarity score has been calculated using the Rissler matrix [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005980#ppat.1005980.ref048" target="_blank">48</a>]. Functionally important residues are highlighted with distinct signs at the bottom of the alignment: cyan triangles for DNA binding and HTH sequestration (R35, R39 and R51); blue triangles for HTH sequestration (E117, E118, E146, D147); green squares for dimerization (R87, D200, K246, E282); red stars for mutated residues (T90, Y91, R92, K100, F171, Y174) and pink stars for other discussed residues (R96, L130, H172, D283, L286). The sequence alignment initially generated by Clustal Ω [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005980#ppat.1005980.ref049" target="_blank">49</a>] displays ComR from <i>S</i>. <i>thermophilus</i> LMD-9 (GenBank ABJ65625.1) and 4 homologues (UniProt numbers) that were experimentally validated as functional for competence activation: <i>S</i>. <i>infantarius</i> (H6PCI7), <i>S</i>. <i>mutans</i> (Q8DWI6), <i>S</i>. <i>pyogenes</i> (Q9A1Y2), <i>S</i>. <i>suis</i> (A4VSD6). The formatting of the multiple alignment has been generated using Espript [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005980#ppat.1005980.ref050" target="_blank">50</a>].</p

Peptide binding mode.

<p><b>A–Electron density map of the bound peptide.</b> The Fo-Fc map, calculated by a simulated annealing protocol without peptide in the structure, is shown contoured at 1σ as a green grid with the peptide in sticks colored by atom type. <b>B–Peptide binding mode.</b> The protein is shown as cartoon colored by spectrum as chain B in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005980#ppat.1005980.g001" target="_blank">Fig 1A</a>. The bound peptide is shown as sticks colored by atom type. Residues K100, T90 and N208 H-bonding with the bound peptide are highlighted in sticks as well as residues F171, H172 and Y174. <b>C–Peptide-binding pocket.</b> ComR is shown as electrostatic surface colored by potential from blue (positive) to red (negative). The bound XIP is shown as sticks colored by atom type and its residues are labeled. <b>D–Schematic drawing of the ComR-XIP interaction</b>. The XIP peptide is shown as purple sticks with blue labels. The polar ComR residues directly interacting with XIP are shown as orange sticks with green labels. Hydrogen bonds are highlighted by green dashed lines. The hydrophobic ComR residues forming the peptide binding pocket are shown as red arcs with spokes radiating towards the ligand. The figure has been generated using Ligplot [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005980#ppat.1005980.ref047" target="_blank">47</a>].</p

DNA binding activity.

<p><b>A–EMSAs performed with ComR mutants affected in dimerization, XIP binding, and activation mechanism</b> (K87A, K246A, K87A/K246A, T90A, Y91A, R92A, K100A, and F171A/Y174A). <b>B–EMSAs performed with ComR mutants affected in HTH sequestration</b> (E117A/E118A and E146A/D147A). EMSAs reactions contain a 40-bp dsDNA encompassing the ComR-box (Cy3-boxP_{<i>ster_1655</i>} wt; 40 ng) and either WT (control) or mutant ComR proteins. Reactions were incubated in the absence (-) or presence of XIP. ComR was used at final concentration of 4 μM, except for mutant ComR(E117A/E118A) where a maximum concentration of 3.5 μM could be reached (*). The gray triangles represent increasing XIP concentrations: 0.2, 0.4, 0.8 and 1.2 μM. The well-defined ComRS-DNA complex and multimeric complexes are respectively indicated by closed and opened arrow heads. One representative experiment of at least 2 independent replicates is shown.</p