Fig. 5: NorA transports drug substrates with 2:1 H⁺:drug stoichiometry.

a Diagram of NorA transport stoichiometries of 2:1 H⁺:ethidium (electrogenic, top) or 1:1 H⁺:ethidium (electroneutral, bottom) and the two energy sources for efflux: proton gradient (∆pH) and membrane potential (∆ψ). Electroneutral transport is driven by ∆pH, while electrogenic transport can be driven by ∆pH or ∆ψ. b Growth inhibition experiments at pH 7.8 of MRSA^∆norA transformed with a hemin-inducible plasmid containing the wild-type norA gene (black circles) or a dead norA mutant control (grey triangles) in the presence of variable ethidium bromide concentration (0 to 125 μg/mL). Bacterial growth is displayed by the OD_600nm value at the 12 hr timepoint. Solid lines correspond to non-linear fits of experimental data to determine the IC₅₀ values. The standard deviations are derived from four replicates obtained from two independent experiments. c IC₅₀ values of plasmids containing wild-type norA divided by the respective control plasmid as a function of different external pH values ranging from 6.5 to 8.5. Mean values ± s.d. are depicted for the IC₅₀ values. d Proposed NorA transport cycle. The NTD and CTD are colored in blue and orange, “S” is the drug substrate, “E222⁻” and “D307⁻” represents deprotonated Glu222 and Asp307, and “E222” and “D307” are protonated Glu222 and Asp307. Step 1: Deprotonation of Glu222 and Asp307 in the inward-occluded state releases the TM5 tether, leading to opening of the substrate binding pocket. Step 2: drug binds to the inward-open conformation of NorA from the cytoplasmic side of the membrane. Step 3: conformational exchange flips the drug-bound inward-open state to the outward-open state. Step 4: drug is released into the periplasm, resulting in the apo, outward-open conformation. Step 5: Glu222 and Asp307 bind protons in the periplasm to give an outward-occluded conformation. Step 6: NorA undergoes conformational exchange to the inward-occluded conformation, preparing it for the next round of substrate turnover.