Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation., Open Access funding enabled and organized by Projekt DEAL., This work was supported by the German Research Foundation via SPP1879 to J.V. and I.H. (VO 1449/1-1 and HA 6322/4-1), and the Heisenberg programme to I.H. (HA 6322/5-1). Research in PJS’s lab is also funded by Wellcome (208361/Z/17/Z) and BBSRC (BB/P01948X/1, BB/R002517/1 and BB/S003339/1). This project made use of time on ARCHER2 and JADE2 granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://hecbiosim.ac.uk), supported by EPSRC (grant no. EP/R029407/1). This project also used Athena and Sulis at HPC Midlands+, which were funded by the EPSRC on grants EP/P020232/1 and EP/T022108/1., Peer reviewed

Supplementary Figures 1-13. -- Supplementary Table 1: Primer used for site-directed mutagenesis in pB24kimA. -- Supplementary Table 2: Cryo-EM data collection, refinement and model statistics. -- Movie 1: Cooperativity Network mobility of CDs in the absence of c-di-AMP. Movie showing the dynamics of selected residues in the KimA cytosolic domain in the absence of c-di-AMP. Movie made over a single 2.2 μs simulation trajectory using VMD, region viewed as in figure 3a. -- Movie 2: Cooperativity Network mobility of CDs in the presence of c-di-AMP. Movie showing the dynamics of selected residues in the KimA cytosolic domain in the presence of two bound c-di-AMP. Movie made over a single 2.2 μs simulation trajectory using VMD, region viewed as in figure 3a. -- Cooperativity Network mobility of CDs in the presence of only one. Movie showing the dynamics of selected residues in the KimA cytosolic domain in the presence of only one bound c-di-AMP. Movie made over a single 0.5 μs simulation trajectory using VMD, region viewed as in figure 3a., Cyclic di-AMP_Supplementary_material.pdf, 41467_2023_38944_MOESM3_ESM.pdf, 41467_2023_38944_MOESM4_ESM.mpg, 41467_2023_38944_MOESM5_ESM.mpg, 41467_2023_38944_MOESM6_ESM.mpg, Peer reviewed