Motile bacteria have the tendency to swim toward the attractant and away from the repellent, the behavior known as chemotaxis. The binding of such molecules by the membrane-bound chemoreceptor results in the modulation of the autophosphorylation activity of CheA, the central kinase in the bacterial chemotactic signaling system. Phosphorylated CheA transfers its phosphoryl group to its cognate response regulators CheY and CheB, which respectively control the direction of flagellar rotation and the level of receptor adaptation, both of which in concert determine the chemotactic response of the cell to the incoming signal.

In this work, we used modern NMR techniques to construct a model for the interaction of the E. coli CheA phosphotransfer domain (P1 domain) and the response regulator CheY in the presence of the CheY-binding domain of CheA, P2. We also investigated the kinetics of phosphotransfer between the P1domain and CheY by stopped-flow fluorescence measurements to further understand the role of the P2 domain and the linker connecting P1 and P2.

Our structural model indicates that the presence of P2, a domain dedicated to binding to the response regulator, leads to a different relative orientation between the phosphotransfer domain and the response regulator from those seen in homologous complex structures previously determined in the absence of a domain equivalent to P2. The kinetics results show that the presence of P2 domain linked to P1 enhances phosphotransfer rate compared the rate by the isolated P1, while the unlinked P2 domain, by itself, is inhibitory to the phosphotransfer. In addition, we obtained evidence that the length of the linker may have been optimized for the phosphorylation of P1 by the kinase domain, P4. Our work contributes to a finer understanding of how these proteins are designed and fine-tuned to meet the biological demand of chemotaxis signaling.