Fig. 3

Salt bridge interaction of charged residues stabilizes extracellular roof architecture. Charged residues in extracellular domains were mutated and transiently expressed in HEK293 cells. a Zoom-in top view shows side chains as ball-and-stick with the positions of negative (red) and positive charges (blue) in the roof. For clarity, ECL1 (gold), ECL2 (old rose), short loop after TMH5 (red), re-entry helix (cyan), ECL3 (violet), TMHs (gray), elbow helix (pink); the intracellular NBD was removed. b Immunoblot detection is shown for ABCG2 mutants using the anti-ABCG2 (BXP-21) antibody in HEK293 cell-free extracts. β-Actin served as an internal loading control. c Quantification of immunoblot is shown for ABCG2 variants (n = 5–17). Data reflect fold-change relative to the WT control. All values are means ± SEM; **P < 0.01; *P < 0.1. Gray dots represent independent biological replicates. d Intracellular localization of the GFP-tagged ABCG2 double mutant R426E E585R in HEK293 cells. The GFP-ABCG2 signal (green) and nuclei (blue) were detected using the Zeiss LSM700 confocal microscope. Scale bar in microscopy images corresponds to 20 µm. e Mitoxantrone efflux activity of ABCG2 mutants expressed in HEK293 cells are shown as percentage relative to WT control (n = 4–9). Gray refer to independent biological replicates. f Conformation of WT, E585R, R426E, and double mutants, taken from the final frames of MD simulations of inward-facing ABCG2, highlighting residues 426 and 585 and the interacting residues on ECL1 (K417) and ECL2 (K500). Dotted lines indicate distances. g Boxplot of distances measured between Cδ of residue 585 and Cζ of residue 426 (boxes indicate second and third quantiles; the center line shows the mean, the dotted whiskers reach the extremes). h Distance boxplot observed between the Cα of residues E585 of the re-entry helix and K500 of ECL2. i Distance boxplot between residue R426 and K417 on ECL1. Data were obtained and merged from three parallel independent simulations over 150 ns for mutants, 500 ns for WT, and shown for both ABCG2 halves at 1 ns space of sampling. The data in g–i were computed based on more than 9000 data points.