Fig. 5: Proton relay network residue D407 is hyper-engaged across protomer states of MdtF.

A Location of four essential proton relay residues (D407, D408, K938, and R969) within the transmembrane domain of RND transporters. B Nile Red efflux assay, performed in PBS buffer (pH 7.4), demonstrates the reduced rate of MdtEF-mediated efflux compared to AcrAB, and the MdtEF^D408A mutant renders the transporter non-functional. E. coli Δ9-Pore cells harbouring p151A-AcrAB, pUC19-MdtEF^WT, pUC19-MdtEF^V610F, or pUC19-MdtEF^D408A plasmids were monitored for fluorescence decay at 650 nm for 200 s. Here, proton gradient-powered efflux pumps were restarted following the addition of 1 mM glucose after 50 s (indicated by the arrow). Reported data are the average and standard deviation from independent measurements (n = 3) and were fitted to a one-phase decay exponential function (Y = (Y₀ − Plateau) * exp(−K * X) + Plateau). C Proton relay residues of MdtF^WT between each of the access, binding, and extrusion states depicted in a cartoon representation. The corresponding density for each residue is depicted in surface form (light pink). D Median H-bond occurrence (in % over the simulation time) at the proton relay site between access, binding, and extrusion states from top to bottom. The circles are indicative of an individual data point, which represents the average hydrogen bond occupancy over each of the three repeated simulations. The bar charts represent the median H-bond occurrence across three repeated simulations. H-bonds were detected using 3.0 Å between acceptor and donor, and an angle smaller than 35° between H, donor, and acceptor (calculated using the cptraj module of Amber24). For K938 and R969, the corresponding residues in MdtF are K940 and R971, respectively. RFU relative fluorescence units.