Fig. 4

The extracellular gate is sealed by two conserved aspartates. a Structure of TM287/288’s extracellular gate in the IF (left, PDB: 4Q4H) and OF (right) state shown as cartoon. D41^TM287and D65^TM288 are shown as sticks and establish hydrogen bonds (dashed yellow lines) with the peptide backbone (shown as sticks) of neighboring helices TM6 and TM6' that are broken during IF–OF transition. b Sequence alignment of bacterial ABC exporters in the region containing the conserved extracellular gate aspartates. c ATPase activities of single mutants D41A^TM287 and D65A^TM288 and the corresponding double mutant (2xDtoA) relative to wild-type TM287/288 determined in detergent. d Drug stimulated ATPase activities of wild-type EfrEF, the single mutants D41A^EfrE and D50A^EfrF and the corresponding double mutant (2xDtoA) reconstituted into proteoliposomes determined in the absence (basal activity) or in the presence of ethidium at the concentrations indicated. Data were normalized to the basal ATPase activity of the respective mutant. The error bars are standard deviations of technical triplicates. e Ethidium accumulation of Lactococcus lactis cells expressing wild-type EfrEF, the inactive Walker B mutant E515Q^EfrF or the extracellular gate mutants D41A^EfrE and D50A^EfrF or the corresponding double mutant (2xDtoA). f DEER analyses probing the extracellular and intracellular TMDs and the NBDs (same positions as in Fig. 2c). DEER traces were recorded in the presence of ATP-EDTA for the wild-type transporter and for TM287/288(2xDtoA). g Relative ATPase activities of the 2xDtoA mutant in the presence of increasing concentrations of Sb_TM#35, Nb_TM#1 and Nb_TM#2. A non-randomized sybody served as control. The data were fitted with a hyperbolic decay function to determine IC₅₀ values as well as residual activities. The error bars are standard deviations of technical triplicates