Asymmetric Hydration and Protonation Switching of Dual Aspartates Drive Flagellar Rotation

Abstract The bacterial flagellar motor is an intricate nanomachine that transforms chemical energy from ion gradients into mechanical rotation, enabling bacterial movement. While stator unit architectures are conserved across species, the molecular link connecting ion translocation to rotational force generation remains elusive. In this study, we refined the cryo-EM structures of MotAB from Campylobacter jejuni (CjMotAB) and integrated a suite of approaches─including single-structure based pKa predictors and free energy perturbation (FEP) calculations, as well as standard and constant-pH molecular dynamics (CpHMD) simulations of various structural models representing the plugged, unplugged, and plug-removed states with different protonation states of D22─to dissect its rotational mechanism. Based on pKa calculations, the D22 residues in chains F and G of MotB were identified as proton carriers supporting the previous hypotheses. Importantly, we observed asymmetric hydration patterns of the two D22 residues in the MotB dimer, along with their hydrogen bonding interactions with MotA T189, which contribute to functional specialization. Our findings reveal that MotA rotation requires two essential prerequisites: plug removal and alternating D22 protonation switching, coupled with dynamic gauche-trans conformational changes in the side chain of D22. This work clarifies how protonation dynamics and structural asymmetry synergistically regulate CjMotAB rotation, advancing our understanding of bacterial flagellar motor function and providing a foundational framework for investigating diverse ion-driven biological motors.