Chemotaxis kinase CheA is regulated by modulating interdomain interactions.

Activation of the chemotaxis kinase CheA, controlled by ligand binding or covalent modification of its associated chemoreceptor, modulates the chemotaxis behavior of bacteria such as Escherichia coli. The activation of CheA, which results in large changes to its autophosphorylation rate, is proposed to involve control of interdomain interactions between the catalytic P4 domain and the substrate P1 domain. While both productive and nonproductive interdomain P1/P4 interactions have been identified and modeled, observation of how these interdomain interactions impact activity has not been determined in functional signaling complexes. Here, we report evidence for changes to CheA P1/P4 interactions upon activation based on changes in chemical shift perturbations of NMR resonances of isolated CheA-P1 domains upon addition of signaling complexes of CheA-P3P4P5, chemoreceptor cytoplasmic fragments, and CheW. We found that CheA-P1 interacts nonproductively with CheA-P4 in kinase-off complexes and forms P1/P1' dimers during this interaction. In contrast, P1 domains in free CheA do not form an observable population of P1/P1' dimers during their nonproductive P1/P4 interaction, so incorporation of CheA into signaling complexes must induce conformational changes to the P4 domain that allow for P1/P1' dimerization and could further stabilize the nonproductive interaction. Activation of CheA in signaling complexes leads to higher affinity interactions between P1 and P4 domains in a productive mode that could lead to higher autophosphorylation activity. The presence of AMPPCP, an ATP analog, focuses the productive interaction into a more specific binding mode that could be necessary for catalysis. Thus, the activation of CheA in signaling complexes is controlled by changes to the P4 domain that control the transient interactions of P1 and P4 domains. Loss of the nonproductive interdomain interaction happens simultaneously with the gain of the high-affinity productive interdomain interaction to activate the kinase CheA.