Dynamic domain arrangement of CheA-CheY complex regulates bacterial thermotaxis, as revealed by NMR

Bacteria utilize thermotaxis signal transduction proteins, including CheA, and CheY, to switch the direction of the cell movement. However, the thermally responsive machinery enabling warm-seeking behavior has not been identified. Here we examined the effects of temperature on the structure and dynamics of the full-length CheA and CheY complex, by NMR. Our studies revealed that the CheA-CheY complex exists in equilibrium between multiple states, including one state that is preferable for the autophosphorylation of CheA, and another state that is preferable for the phosphotransfer from CheA to CheY. With increasing temperature, the equilibrium shifts toward the latter state. The temperature-dependent population shift of the dynamic domain arrangement of the CheA-CheY complex induced changes in the concentrations of phosphorylated CheY that are comparable to those induced by chemical attractants or repellents. Therefore, the dynamic domain arrangement of the CheA-CheY complex functions as the primary thermally responsive machinery in warm-seeking behavior.

change induced by the P2 domain-CheY interaction, we created CheY mutants with decreased affinities for the P1 domain, and examined the CheY-induced chemical shift perturbation of the mutants. An alanine mutation was introduced into I20 of CheY, which is close to D57 and sufficiently distant from the P2 domain, in the previously reported structure of the isolated P2 domain-CheY complex 37 , and the affinities of the CheY/I20A mutant for the full-length CheA and the isolated P2 domain were determined by isothermal titration calorimetry. As a result, the association constant of the CheY/I20A mutant for the full-length CheA was significantly lower than that of the wild type, whereas the affinity of the CheY/I20A mutant for the P2 domain was almost identical to that of the wild type    Fig. S4a-c). The CheA-CheY interaction was also perturbed by introducing mutations of other residues of CheY, including N59K. Therefore, the CheY-induced chemical shift perturbation observed for the resonances from the P1 domain in the full-length CheA is derived from the P1 domain-CheY interaction.

Interaction between the isolated P1 domain and CheY
In the experiments using the isolated P1 domain ( Supplementary Fig. S4d) S4g).

CheY autophosphorylation reactions
Ligand binding and methylation of the chemoreceptors affect the autophosphorylation rates of the CheA-chemoreceptor complexes, which determine the concentration of the phosphorylated CheA (CheA-P), and it is possible that the autophosphorylation rates are different among cells that express only one of the chemoreceptors. In the case of a low CheA-P concentration, the amount of phosphorylated CheY (CheY-P) decreases with increasing temperature, due to the decrease of the P1-CheY unbound state, in which autophosphorylation occurs ( Supplementary Fig. S9a). The decrease of CheY-P leads to the motion to the higher temperature (warm-seeking behavior). In contrast, under the conditions with high CheA-P concentrations, CheY-P increases at higher temperatures, due to the increase of the P1-CheY bound state, in which phosphotransfer occurs. The decrease of CheY-P leads to the motion to the cold temperature (cold-seeking behavior).
Although the aforementioned schemes are complicated by the phosphotransfer from CheA to CheB and the demethylation of the chemoreceptors by the phosphorylated CheB, our quantitative time-course calculation of the signaling demonstrated that the sign of the temperature-dependent change of the CheY-P concentration is perturbed by the attractant binding, and its sign can be switched by the complete methylation of the chemoreceptor 7 ( Supplementary Fig. S9b). Therefore, the temperature-dependent shift of the dynamic domain arrangement of the CheA-CheY complex explains the effects of the chemoreceptors on the thermotaxis.   Fig. S3a-d).
Data represent the mean ± SE mean of two, three, or four separate experiments. *P < 0.05, unpaired one-tailed Student t test.