Extended Data Fig. 6: Functional and biochemical analysis of the LetA polar network and ZnR domains, related to Fig. 3.

a, Western blot analysis showing results of pull-down assay, used to assess the interaction between LetA polar network mutants and LetB. His-LetA WT and mutants were used as the bait for pull-down, and interaction with untagged LetB was assessed using α-LetA (clone 72) and α-LetB antibodies. LetA (WT or mutants) and WT LetB are co-expressed from the same plasmid. Three independent biological replicates were performed starting with three different colonies, on different days, with similar results. b, Water density map from equilibrium MD simulations for LetA. The map was generated using water oxygens within 3.5 Å of LetA and averaged over all the frames in the equilibrium MD simulations (-Lipid 1). LetA is shown in cartoon representation, where the helices are depicted as cylinders. TMD^N and TMD^C are colored according to Fig. 2a. The water density is colored in light yellow, except for the densities inside TMD^C, which are highlighted in orange. c, Hydrogen-bond network between proposed proton shuttle residues and water. The most frequently occurring water bridges in the equilibrium MD simulations (-Lipid 1, three replicas) are shown. Each line represents the water bridge that most consistently appears between the two corresponding nodes throughout the simulation. Each polar residue is shown as a node, with the lines connecting the nodes representing water bridges. The thickness of the lines corresponds to the relative occupancy of the water bridge throughout the simulation. The color coding of the edges indicates the number of water molecules involved in forming the bridge: 1-W (green) represents a single water molecule bridge, 2-W (blue) indicates a bridge formed through two water molecules, and 3-W (orange) shows a water bridge through three water molecules. The percentage occupancy of each water bridge is annotated alongside the edges. d, Cellular assay to examine the function of LetA ZnR cysteine mutants. 10-fold serial dilutions of the indicated strains were spotted on LB agar with or without cholate or LSB. All strains are constructed in a ΔpqiAB background. e, Western blot analysis of lysates of the strains indicated, to compare cellular levels of WT LetA and ZnR cysteine mutants. α-LetA (clone 72) was used to probe LetA. BamA levels were probed using an α-BamA antibody as a loading control. Three independent biological replicates were performed starting with three different colonies, on different days, with similar results. f, Sequence alignment of the LetA and PqiA ZnR domains. g, Cellular assay to assess the function of LetA mutants, in which the ZnR domains of LetA are replaced with the ZnR domains from E. coli PqiA. WT LetB is co-expressed with each ZnR mutant. 10-fold serial dilutions of strains were spotted on LB agar with or without the indicated detergent. All strains are constructed in a ΔpqiAB background. h, Western blot analysis of lysates of the strains indicated, to compare cellular levels of WT LetA and a ZnR mutant in which the ZnR domains in LetA are swapped with those of the E. coli PqiA protein. α-LetA (clone 72) was used to probe LetA. BamA levels were probed using an α-BamA antibody as a loading control. Three independent biological replicates were performed starting with three different colonies, on different days, with similar results. i, Cellular assay to assess the function of LetA mutants, in which the ZnR domains of LetA are deleted. WT LetB is co-expressed with each ZnR mutant. 10-fold serial dilutions of strains were spotted on LB agar with or without the indicated detergent. All strains are constructed in a ΔpqiAB background. j, Western blot analysis of lysates of the strains indicated, to compare cellular levels of WT LetA and ZnR deletion mutants. Anti-LetA (clone 72) was used to probe LetA. BamA levels were probed using an anti-BamA antibody as a loading control. Three independent biological replicates were performed starting with three different colonies, on different days, with similar results. k, Western blot analysis showing results of pull-down assay from purified membrane fractions, used to assess the interaction between LetA ZnR deletion mutants and LetB. His-LetA WT and mutants were used as the bait for pull-down, and interaction with untagged LetB was assessed using α-LetA (clone 72) and α-LetB antibodies. Three independent biological replicates were performed starting with three different colonies, on different days, with similar results. Gel source data for panels (a), (e), (h), (j), and (k) are provided in Supplementary Fig. 1h,i,j,k,l, respectively.