Fig 1.
Sequence conservation between ABCB1 and ABCB11 mapped onto a structural model of ABCB1.
The NBD in front is shown in green ribbon representation, while the NBD in the back shows the NBD interface in surface rendering. The surface is colored according to sequence similarity between ABCB1 and ABCB11. Blue color indicates residue identity, orange indicates maximal residue divergence. The four differing residues in NBS1 are highlighted in magenta.
Fig 2.
UIC2 binding to ABCB1 variants carrying mutations in NBS1.
(A) Total cell surface ABCB1 expression levels were determined using the conformation insensitive 15D3 anti-ABCB1 antibody (means ± SD of n = 5 independent experiments). (B) Changes of UIC2 reactivity in wild-type ABCB1 (orange), the single (E556M, green) and the quadruple (E556M/S474E/G1178R/Q1180E, blue) mutant in response to cyclosporin A (CsA, 10 μM) treatment or ATP depletion. NIH 3T3 cells expressing the ABCB1 variants were labeled with Alexa 647 conjugated UIC2 or 15D3 mAbs (with comparable dye to antibody ratio (F/P)). The percentage of UIC2-bound ABCB1 conformers was calculated relative to the total cell surface ABCB1 expression levels determined by 15D3 labeling (mean ± SD of n = 5 independent experiments). Significant differences compared to untreated controls or wild-type are shown by *** (p<0.001).
Fig 3.
NIH 3T3 cells expressing ABCB1 variants were permeabilized to allow for systematic variation of intracellular nucleotide concentrations. Dose-response curves of UIC2 binding with increasing concentrations of MgATP in the absence or presence of vanadate (Vi) were obtained for (A) wild-type, (B) the catalytic glutamate mutant and (C) the quadruple mutant. Dose-response curves were normalized to the UIC2 signal obtained in the presence of 0 mM ATP, which was found practically equal to the F/P corrected 15D3 signal and did not differ between the different ABCB1 variants. Panel (D) summarizes the mean apparent nucleotide affinity values (KA ± SD, n = 9–11) obtained in control and Vi treated cells. Statistical comparison of the KA values of Vi treated and untreated samples was carried out by unpaired Student's t-test (two-tailed test); ***p<0.001, **p<0.01.
Fig 4.
Effect of the mutations on substrate-stimulated steady state ATPase activity.
(A) Expression levels of the ABCB1 variants in membranes isolated from NIH 3T3 cells, detected by Western blotting using the G-1 anti-ABCB1 mAb. (B) Steady state ATPase activity of ABCB1 variants in membranes isolated from NIH 3T3 cells. Membranes were incubated for 25 min in the presence of 3 mM MgATP and the indicated concentrations of verapamil. ABCB1 specific steady state ATPase activity is defined as valspodar inhibitable vanadate sensitive ATPase activity. Mean ± SD of 5 independent experiments are shown. (Significant differences compared to untreated NIH 3T3 control membrane samples are shown by ***: P<0.001, **: P<0.01, *: P<0.05, while significant differences between the verapamil treated and untreated samples are indicated by †††: P<0.001, ††: P<0.01, †: P<0.05). (C) Response of the ABCB1-specific steady state ATPase activities to verapamil stimulation. Significant differences between the wild-type and the quadruple mutant ABCB1 are shown by **: P<0.01.
Fig 5.
Transport activity of ABCB1 variants.
ABCB1 dependent (A) Hoechst 33342 (B) and calcein-AM transport were quantified by the transport activity factor (TAF). NIH 3T3 cells expressing ABCB1 variants were incubated with Hoechst 33342 (0.025, 0.1, 0.5, 1 and 5 μM, panel A) or calcein-AM (0.025, 0.05, 0.1, 0.25, and 0.5 μM, panel B) for 30 min at 37 oC. Mean ± SD of three independent experiments are shown. Significant differences compared to ABCB1-negative cells: ***: P<0.001, **: P<0.01.
Fig 6.
(A) Distribution of distances between the Cα atom of residue S434 (Walker A) and the Pα of ATP. (B) Time evolution of the RMSD of ATP in NBS1 after fitting to NBD1. Slight variations in color are used to distinguish the three replicates. (C) Zoom into NBS1 showing the binding mode of ATP. (D) Distribution of distances between the Cα atoms of residue S434 (Walker A) and residue G/R1178 (signature sequence). (E) Distribution of distances between the Cα atoms of residue S1177 (signature sequence) and the Pα of ATP. Data from independent trajectories for wild-type simulations are shown in orange, the trajectories of the catalytic glutamate mutant are shown in green, and data for the quadruple mutant are shown in blue.
Fig 7.
Structural changes in the NBD interface associated with NBS1 mutations.
(A) Two dimensional probability distribution of distances: the x-axis reports on the distance between the signature sequences of NBD1 and NBD2, the y-axis shows the distance across NBS1 between the Walker A motif (residue 434) and the signature sequence in NBS1 (residue 1178). (B) Two dimensional probability distribution of the distance within NBS1: the x-axis reports on distances between the signature sequences of NBD1 and NBD2, the y-axis measures distance within NBS1 between the Q-loop of NBD1 (residue 475 and 476) and the signature sequence in NBS1 (residue 1178). (C) Probability scale for panel A and B. (D) Visualization of the electrostatic potential (calculated using APBS without the presence of ATP and Mg2+) in a plane perpendicular to the main axis of ABCB1 and intersecting with both signature motifs. The electrostatic potential was averaged over the replicates of wild-type ABCB1 simulations (E) Electrostatic potential of the quadruple mutant as determined in panel C.
Fig 8.
The guanidinium moiety of G1178R interacts with both Q-loops.
(A) Distribution of distances between the Q-loop carbonyl oxygens of Q475 and Q1118. Data from independent trajectories for wild type simulations are shown in orange, the trajectories of the catalytic glutamate mutant are shown in green, and data for the quadruple mutant are shown in blue. (B) Time evolution of distances between the Cα atoms of the D-loop serines S559 and S1204. (C) Relative D-loop orientation shown for wild-type ABCB1, highlighted by D-loop serines S559 and S1204 (magenta). (D) Representative position of the guanidinium group of G1178R (cyan) forming hydrogen bonds with the backbone carbonyl of Q-loop glutamines (yellow).