Insights into Chemoreceptor MCP2201-Sensing D-Malate

Abstract

Bacterial chemoreceptors sense extracellular stimuli and drive bacteria toward a beneficial environment or away from harm. Their ligand-binding domains (LBDs) are highly diverse in terms of sequence and structure, and their ligands cover various chemical molecules that could serve as nitrogen, carbon, and energy sources. The mechanism of how this diverse range of LBDs senses different ligands is essential to signal transduction. Previously, we reported that the chemoreceptor MCP2201 from Comamonas testosteroni CNB-1 sensed citrate and L-malate, altered the ligand-free monomer-dimer equilibrium of LBD to citrate-bound monomer (with limited monomer) and L-malate-bound dimer, and triggered positive and negative chemotactic responses. Here, we present our findings, showing that D-malate binds to MCP2201, induces LBD dimerization, and triggers the chemorepellent response exactly as L-malate did. A single site mutation, T105A, can alter the D-malate-bound LBD dimer into a monomer-dimer equilibrium and switch the negative chemotactic response to D-malate to a positive one. Differences in attractant-bound LBD oligomerization, such as citrate-bound wildtype LBD monomer and D-malate-bound T105A dimer, indicated that LBD oligomerization is a consequence of signal transduction instead of a trigger. Our study expands our knowledge of chemoreceptor-sensing ligands and provides insight into the evolution of bacterial chemoreceptors.

Keywords: chemoattractant; chemoreceptor; chemorepellent; enantiomer.

Figure 1

MCP2201 senses D-malate and triggers a chemorepellent response. (A) D-malate affinity measured by an isothermal titration calorimeter. (B) The chemotactic responses of CNB-1Δ20 cells harboring MCP2201, or not, toward D-malate (left) and L-malate (right) on the gradient soft-agar plate. (C) Chemotactic responses of MCP2201 toward different concentrations of D-malate (blue) and L-malate (green); Δ20 (pink) responds to different concentrations of D-malate in the capillary assays. Data were fitted to a logistic function. The experiments were repeated three times. The representative example is shown in (A,B).

Figure 2

D-malate binds to the same pocket as L-malate, yet is subtly different. (A) D-malate-bound LBD dimer and the ligand binding pocket. (B) Superposition of D-malate (orange and green) and L-malate-binding pockets (blue and pink, PDB accession code 7WRM). Hydrogen bonds in the D-malate- and L-malate-bound structures are shown in yellow and purple, respectively, with binding motifs highlighted in green and pink. (C) Diagram comparison of D-malate (left) and L-malate (right) binding motifs.

Figure 3

D-malate induces LBD dimerization as L-malate. (A) D-malate-bound dimeric interface. The two subunits are colored in light and dark colors. (B) Analytical ultracentrifugation assays of ligand-free, L-malate-bound, and D-malate-bound MCP2201 LBD. (C) ITC assays of MCP2201 LBD oligomer dissociation in the presence of D-malate. Calorimetric dilution data (top) for injection of MCP2201 LBD in the presence of 10 mM D-malate at 25 °C were integrated, and dilution-corrected peaks were fitted to an oligomer dissociation model (bottom) to assess the dissociation constants.

Figure 4

Chemotactic responses of MCP2201 mutants to D-malate. (A) Chemotactic responses of CNB-1Δ20 cells harboring MCP2201 mutants to L-malate, D-malate, and citrate in the capillary assay. Data are presented as mean ± SD (n = 6 biological replicates). Statistical significance was determined by an unpaired one-tailed Student’s t-test. ** (p < 0.01), and **** (p < 0.001) showed a significant difference between ligand-treated and non-treated groups. The “ns” stands for not significant. (B,C) D-malate (B) and citrate (C) affinity of T105A determined by isothermal titration calorimetry assays. (D) Chemotactic responses of Δ20 cells harboring T105A (green), or not (pink), toward different concentrations of D-malate in the capillary assay. Data were fitted into the logistic function. (E) Analytical ultracentrifugation assays of ligand-free, D-malate-bound, and citrate-bound T105A LBD. The experiments were repeated three times, and the representative instance is shown here.

Figure 5

The relative abundances of alanine substitution of T105 in MCP2201 LBD similarities and different MCP2201 orthologs. The relative abundances of different substitutions of T105 in the top 1–100, 101–250, 251–500, 501–1001, and 1001–3349 sequences of LBD similarities and previously reported 201 MCP2201 orthologs are shown in a histogram.