Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites

Abstract

Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100- to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220- to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed that the ligand-binding sites could be superimposed and that amino acids involved in ligand recognition are conserved in both binding sites. Ligand binding to both modules was shown to trigger chemotactic responses. Further analysis showed that McpS-like receptors were found in different classes of proteobacteria, indicating that this mode of response to different carbon sources may be universally distributed. The physiological relevance of the McpS architecture may lie in its capacity to respond with high sensitivity to the preferred carbon sources malate and succinate and, at the same time, mediate lower sensitivity responses to the less preferred but very abundant carbon source acetate.

Fig. 1.

Three-dimensional structure of the LBR of the McpS chemoreceptor. Ribbon representation in which each monomer is colored differently. The helices and loops are numbered. The binding sites for malate, succinate, and acetate are indicated.

Fig. 2.

Superimposition of the proximal module with the distal module of McpS-LBR. This alignment was made with the DALI pairwise alignment algorithm. Bound acetate is shown in blue and bound malate in green. Red, distal module; yellow, proximal module.

Fig. 3.

Structural basis for ligand recognition at McpS-LBR. (A) Representative section of the final 2F_o−F_c electron density map including data from 25 to 1.8 Å contoured at 2.5 σ above the mean of the map. The region shown corresponds to the malate-binding site with a molecule of malate shown. (B and C) View of the interaction of malate (B) and succinate (C) with the protein. Q65 is from the A monomer, whereas the remaining interactions are with the B monomer. (D) View of the interaction of acetate with McpS-LBR. (E) Superimposition of structures containing malate (green) and succinate (orange).

Fig. 4.

Microcalorimetric titrations of native and mutant McpS-LBR with malate. (Upper) Raw titration data of McpS-LBR, McpS-LBR R60A, and McpS-LBR R254A. Protein was at 33 μM, and aliquots of 1 mM malate were injected. (Lower) Integrated, concentration-normalized, and dilution heat-corrected raw data of the titration of McpS-LBR with malate.

Fig. 5.

Quantitative capillary chemotaxis assay of P. putida strains toward alanine (A) and acetate (B). Data are means of at least three independent experiments, each conducted in triplicate. The average number of cells that swam into the capillary has been subtracted.

Fig. 6.

Terminal helix α6 is the proposed signaling helix. Shown is a monomer of McpS-LBR and helix α6, proposed to be the signaling helix (highlighted). Bound malate and acetate are shown in orange and yellow, respectively. R254, T258, and Y236, involved in ligand binding, are shown in stick mode.