BPS2026 – Molecular simulations of CheA conformational dynamics and ATP binding in bacterial chemotaxis

Sensory signal transduction in bacterial chemotaxis is mediated by core-signaling units (CSUs) composed of the histidine kinase CheA, transmembrane chemoreceptors, and the adaptor protein CheW. By modulating CheA autophosphorylation in response to chemoreceptor ligand binding, CSUs regulate downstream response regulators that control flagellar rotation and cellular motility. Despite its essential role in chemotaxis, the molecular basis of CheA regulation remains incompletely understood, owing to its flexible, multi-domain architecture and large-scale conformational transitions. To help address this, we used AlphaFold3 to predict intact CSU baseplate models with and without ATP. The resulting structures reveal pronounced differences, especially in the localization of two key CheA domains: (1) the P4 catalytic domain, which binds and hydrolyzes ATP and (2) the P1 phosphotransfer domain, which transfers a phosphoryl group from ATP to P2 and subsequently to the response regulator. The most striking of these differences is the prediction that P4 adopts a previously identified (yet unpublished) “dipped” conformation in the presence of ATP. Based on the location and orientation of the substrate histidine relative to the ATP-binding site, we constructed putative models of the kinase-ON and kinase-OFF CSU signaling states, which we subject to multi-microsecond atomistic molecular dynamics (MD) simulations. Our simulations reveal that the P1 domain greatly suppresses the conformational dynamics of neighboring domains through specific interfacial contacts in the kinase-OFF state, stabilizing CheA overall. Additionally, in the kinase-ON state, our simulations show that ATP binds tightly to the dipped P4 conformation and forms dynamic, stabilizing interactions with the ATP-lid, suggesting this conformation may tune both ATP binding affinity and the dynamic coupling between P1 and P4 during activation. Taken together, our findings provide fresh mechanistic insights into CheA signaling, highlighting how specific interdomain interactions and ATP binding shape kinase activity.