HDX-MS reveals that bacterial chemoreceptor signaling complexes control kinase activity by stabilizing the catalytic domain of cheA.

Abstract

Although bacterial chemotaxis is one of the best-understood signaling systems, it remains unclear how chemoreceptors regulate the activity of the associated histidine kinase CheA. A signaling complex comprises of two trimer-of-dimers of membrane-bound chemoreceptors, two CheW coupling proteins, and dimeric CheA. We have used hydrogen deuterium exchange mass spectrometry (HDX-MS) to reveal how E.coli CheA domain structure and dynamics change within signaling complexes during kinase activation and inhibition. CheA is a five-domain homodimer: P1 bears a phospho-accepting His48, P2 binds downstream effector proteins, P3 mediates dimerization, P4 is the catalytic domain, and P5 binds CheW and the chemoreceptor. We assemble native-like complexes of CheA with CheW and the cytoplasmic fragment (CF) of the aspartate receptor bound to vesicles. Kinase-off states (free CheA and CheA in complexes with unmethylated CF) exhibit rapid deuterium uptake in P4. In contrast the kinase-on state (CheA in complex with methylated CF) exhibits significantly slower deuterium uptake in P4, suggesting that stabilization and destabilization of P4 is key to kinase activation and inhibition. P3 exhibits faster deuterium uptake in complexes with the unmethylated receptor, suggesting that the mechanism of adaptation involves destabilization of P3. We propose this destabilization is due to increased interactions between P3 and unmethylated CF. Cysteine crosslinking will test whether P3-chemoreceptor crosslinking differs between kinase-off and kinase-on complexes. The very slow exchange of two helices in P1 in all states identifies the dimerization interface of the P1/P1’. Cysteine crosslinking will determine whether P1 forms a symmetric or antisymmetric dimer. It's clear that chemotaxis is a significant factor in pathogenesis. Our results provide insight into the mechanism of CheA kinase control that may aid in the development of novel antibiotics. This research supported by NIH R01 GM120195.