Fig. 3: V610F creates a hydrophobic nook within the DBP of MdtF.

A Location of the V610F residue observed within our cryo-EM structure for MdtF^WT. The switch loop and V610 residue is indicated in blue and red, respectively. The residue is found immediately upstream of the switch loop and protrudes into the DBP between the PC1 and PN2 subdomains. B MIC calculations to demonstrate the altered MDR phenotype because of the V610F mutation within MdtF. The values and log-fold change values are displayed to demonstrate the relative effects within E. coli Δ9-Pore cells. C Structure of apo-MdtF^V610F solved by cryo-EM at a resolution of 3.28 Å with an observed formation of a ‘hydrophobic nook’ within the DBP, arising due to the single-point mutation. D Binding of R6G by MdtF^WT and MdtF^V610F as determined by a fluorescence polarisation assay. R6G was maintained at 1 μM throughout, and its emission wavelength was 550 nm. The binding isotherms demonstrate a K_D of 0.39 ± 0.09 and 0.72 ± 0.09 µM for MdtF^WT and MdtF^V610F, respectively, in a buffer containing 50 mM sodium phosphate, 150 mM NaCl, 10% glycerol. Reported data are the average and standard deviation from independent measurements (n = 3) and were fitted to a hyperbola function (FP = (Bmax * [protein])/(K_D  +  [protein])). E Structure of R6G-MdtF solved by cryo-EM at a resolution of 3.20 Å, demonstrating cation-π (R6G-Phe626) and π-π (R6G-Phe178) interactions with R6G in the hydrophobic binding pocket of MdtF (colouring on the molecular surface from dark cyan (most hydrophilic) to white to dark golden (most lipophilic)). F MdtF^V610F demonstrates a more open cleft within the binding pocket compared to MdtF^WT. R6G binding is facilitated by a reorientation of the Phe626 residue. DBP distal binding pocket, FP fluorescence polarisation, MIC minimum inhibitory concentration, R6G Rhodamine 6G.