CheA-receptor interaction sites in bacterial chemotaxis

Abstract

In bacterial chemotaxis, transmembrane chemoreceptors, the CheA histidine kinase, and the CheW coupling protein assemble into signaling complexes that allow bacteria to modulate their swimming behavior in response to environmental stimuli. Among the protein-protein interactions in the ternary complex, CheA-CheW and CheW-receptor interactions were studied previously, whereas CheA-receptor interaction has been less investigated. Here, we characterize the CheA-receptor interaction in Thermotoga maritima by NMR spectroscopy and validate the identified receptor binding site of CheA in Escherichia coli chemotaxis. We find that CheA interacts with a chemoreceptor in a manner similar to that of CheW, and the receptor binding site of CheA's regulatory domain is homologous to that of CheW. Collectively, the receptor binding sites in the CheA-CheW complex suggest that conformational changes in CheA are required for assembly of the CheA-CheW-receptor ternary complex and CheA activation.

Fig. 1

NMR analysis of the bindings of unlabeled CheAΔ354 and CheW to ¹³C-methyl labeled TM0014_90-206. (a) Methyl TROSY sections (left, Ile region; right, Leu region) of TM0014_90-206 (150 μM) in the presence (red, at 1:4 ratio of TM0014_90-206:CheAΔ354) and absence (black) of CheAΔ354. Residues with significant chemical shift changes are numbered with the arrows indicating the direction of the peak movement. (b) Same Sections of TM0014_90-206 (150 μM) spectra with (red, at 1:4 ratio of TM0014_90-206: CheW) and without (black) CheW. (c) Same sections of TM0014_90-206 (150 μM) spectra before (black) and after (red, at 1:4 ratio of TM0014_90-206:CheAΔ354-CheW) the addition of CheAΔ354-CheW complex. (d) Residues showing significant chemical shift changes are mapped onto the structure of TM0014 dimer (one monomer shown in cartoon and the other in ribbon format; PDB ID: 3G67).

Fig. 2

NMR and mutational analysis of the receptor binding site of CheA. (a) Methyl TROSY section of ¹³C-methyl labeled CheAΔ354 (150 μM) in the presence (red, at 1:4 ratio of CheAΔ354:TM0014_90-206) and absence (black) of TM0014_90-206. Residues with significant chemical shift changes are numbered with the arrows indicating the direction of the peak movement. (b) Same section of the spectra of methyl-labeled CheAΔ354 (150 μM) in complex with unlabeled CheW before (black) and after (red, 1:4 ratio of CheAΔ354-CheW: TM0014_90-206) the addition of TM0014_90-206. (c) Residues (red) showing significant chemical shift changes are mapped onto the structure of the P5 domain of CheA (PDB ID: 1B3Q). The region involved in CheW binding is colored in green. (d) Structural alignment of CheW (yellow, PDB ID: 1K0S) and the P5 domain of CheA (grey). The two residues (Val27 and Val98) that show the largest shifts in the CheW-receptor interaction are highlighted in blue, and Ile563 and Ile566 that shift upon receptor binding are in red. (e) ATPase assays (s.d., n=3) for the basal autophosphorylation activities, (f) in vitro CheA activation assays (s.d., n=3) for the activation abilities, and (g) in vivo swarm assays for the chemotactic abilities of the wild-type E. coli CheA and S534R mutant (duplicate) at the receptor binding site of the P5 domain determined by NMR.

Fig. 3

Receptor binding sites of the CheA–CheW complex. The binding sites determined by NMR are shown in red.