Structural effects of the R482 variations.

<p>The R482G mutation is able to alter the positioning of TM helices and the conformation of the P480 kink. Two structures were taken from the end of two simulations, which exhibited the largest changes, to decipher and demonstrate the effect of R482. The distances between Cα of R482 (TH3) and that of Q398 (TH1), S441 (TH2), and A517 (TH4) were measured throughout the simulation trajectory, and in the last frame exhibited the following values: distances of A, B, and C in WT are 8.4 Å, 7.2 Å, and 7.4 Å, while in the R482G variant are 15.1 Å, 8.1 Å, and 4.9 Å, respectively. The right panels contain both the WT and R482 structures in cylindrical representation. Arrows are placed at spots, which exhibit the most pronounced differences between the two constructs, and point from the wild type to the mutant conformation. TH1-6 are colored by red, green, blue, orange, magenta, and yellow, respectively.</p

<i>In silico</i> docking shows binding sites along a potential substrate pathway.

<p>Substrates and non-substrates were docked to six ABCG2 conformations. Both types of molecules could dock at Site 1 (blue), while only the binding of substrates could be observed at Site 2 (red). The central Site 3 (yellow) resides between the two monomers. A potential off-site at the extracellular part is also revealed (Site 4, magenta). Here, docking poses of sulfasalazine are shown in the case of two ABCG2 conformations (two out of the last frames of the six equilibrations are shown).</p

The general structural properties of the ABCG2 homology model.

<p>The two monomers are colored by different light green colors. The most important parts, providing the interface between the TMD and NBD are the coupling helix (light blue) and the connecting helix (dark green). The functionally important R482 is colored ruby. The site of the most frequent polymorphism, Q141 is deep purple. The location of important mutations affecting biogenesis and function are labeled by dark green (R383) and orange (K86), respectively. Residues, which are probably significant in cholesterol modulation, are blue (Y413) and magenta (a.a. 555–558). Gray dots represent the boundaries of the hydrophobic region of the bilayer, defined by the OPM webserver. <b>Insert:</b> ABCG2 (green) and mouse ABCB1/Pgp (blue, PDBID: 4M1M) are overlaid. The mouse ABCB1 NBD is much further from the membrane bilayer and the distance between the NBDs of ABCB1 and ABCG2 (the Cα atoms of the Walker A Lys residues; K433 and K86, respectively) is 26 Å.</p

The Q141K variant interferes with the coupling between the NBD and the connecting helix.

<p>The side chain of F142, which is at a homologous position as the CFTR F508 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164426#pone.0164426.s002" target="_blank">S2 Fig</a>), is clamped by the positively charged K382 and R383. The positive charge of 141K destabilizes this interaction by repulsion with K382, as shown by molecular dynamics simulations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164426#pone.0164426.s005" target="_blank">S5 Fig</a>).</p

The structural background of cholesterol regulation.

<p>The last frame of a 50 ns long MD simulation with ABCG2 embedded in a POPC bilayer shows that the CRAC motif, containing Y413, is located in the charged area of POPC head groups (orange), as a rational location for cholesterol biding. Also, the leucine based cholesterol binding motif (magenta) is situated in this layer. Right panel: zoomed area reveals a close contact between the CRAC and the leucine based motives (e.g. Y413 and V556 are closer than 5 Å) and may provide a cholesterol binding site (black circle). Gray: POPC hydrophobic tails; orange: charged head groups of lipids.</p

Effect of diffusion rate and ABC₀ affinity to drug on the level of network components.

<p>Time course simulations were run from steady states belonging to different parameter sets as described in Methods. The extracellular drug concentration ([X_e]) was set to 75 nM at t₀ = 0 h. Parameter values are expressed as multiples of their default value (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115533#pone.0115533.s007" target="_blank">S3 Table</a>). Concentration profile of X_c, X’_c, X”_c and the level of ABC₀ were plotted. <b>a-d</b> Effect of diffusion rate through membranes. Five simulations were run by setting the diffusion rate constants to different values of four orders of magnitude. <b>e-h</b> Effect of ABC₀ affinity to drug (K_m). Five simulations were run by setting the Michaelis constant to different values encompassing four orders of magnitude.</p

Simplified wiring diagram of the chemoimmune network model.

<p>Modeled interactions of a single xenobiotic (X) with Phase 0-III effector enzymes and regulators. Solid arrows represent transport through membranes or biochemical reactions. Dashed arrows denote regulation including multi-step transcriptional and translational regulation (gray) and more direct interaction (black), such as binding of a drug to nuclear receptors. ABC₀ and ABC_III symbolize general Phase 0 and Phase III efflux transporters, respectively. CYP and GST represent a Phase I oxidase (a member of the cytochrome P450 superfamily) and a Phase II GSH transferase, respectively. NR symbolizes a general xenobiotic nuclear receptor, while Nrf2 denotes a specific transcription factor. (GST, ABC_III and Nrf2 are duplicated to increase clarity of the figure. Regulatory arrows are not duplicated.) Letters ‘c’ and ‘e’ indicate cytoplasmic and extracellular localization, respectively. X’_c is the CYP-oxidized cytoplasmic metabolite of X_c. X”_c is the glutathione-conjugated form of X’_c. X’_bc represents reactive species produced by normal cell metabolism. X’_bc is metabolized by the same pathway as X’_c. Negative feedback loops are X_c → NR → CYP —| X_c, X’_c (and X’_bc) → Nrf2 → GST —| X’_c (and X’_bc), X_c → NR → ABC₀ —| X_c, X_c → NR → Nrf2 → ABC₀ —| X_c, where → denotes activation and —| denotes inhibition. Feedforward loops are X_c → NR → GST —| X’_c, X_c → NR → ABC_III —| X”_c (‘direct’ regulation) and X_c → NR → Nrf2 → GST —| X’_c, X_c → NR → Nrf2 → ABC_III —| X”_c (‘indirect’ regulation). For the complete wiring diagram with all details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115533#pone.0115533.s001" target="_blank">S1 Fig.</a></p

<i>In silico</i> cytotoxicity curves reveal impact of drug’s toxicity profile and protein level on survival.

<p>Time course simulations were run for up to 48 hours from steady states belonging to different parameter sets and extracellular drug concentrations ([X_e], set at t₀ = 0 h) as described in Methods. The minimal <i>Fitness</i> values reached in simulations were plotted against [X_e] (colored circles; connected by interpolation curves—see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115533#pone.0115533.s011" target="_blank">S1 Text</a>). Critical concentrations of X’_c and X”_c are constant on all panels with values 1 μM and 100 μM, respectively. Critical concentration of X_c ([X_c,crit]) is indicated on the panels. <b>a</b> Impact of the toxicity of the unmetabolized form of the drug ([X_c,crit]) on <i>in silico</i> cytotoxicity. <i>In silico</i> cytotoxicity curves were plotted for [X_c,crit] values from 0.05 nM to 5 μM. The EC₅₀ value is indicated for [X_c,crit] = 0.5 nM. <b>b</b> Impact of transporter and oxidase levels on <i>in silico</i> cytotoxicity when X_c is more toxic than X’_c ([X_c,crit] = 0.05 nM). <b>c</b> Impact of transporter and oxidase levels on <i>in silico</i> cytotoxicity when X_c is less toxic than X’_c ([X_c,crit] = 5 μM). In panels <b>b</b> and <b>c</b><i>in silico</i> cytotoxicity curves were calculated after setting the transcription rates of ABC₀ or CYP 0.2 or 5 times of its original value (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115533#pone.0115533.s007" target="_blank">S3 Table</a>) to model lower or higher expression levels.</p

Modeling cellular fitness to study cytotoxic effects.

<p>Calculation of cellular fitness by assuming a single toxic compound, X. Cytoplasmic concentration of the drug ([X_c]) was calculated using the time course simulation described in Methods. <i>Critical concentration</i> of X_c ([X_c,crit], dashed yellow line) was defined as the threshold concentration, which must be exceeded to cause cellular damage. <i>Chemical load</i> is defined as a nonlinear function of the [X_c]/[X_c,crit] ratio (magenta curve). The cell is assumed to have a constant <i>Regeneration capacity</i> (dashed black line). When <i>Chemical load</i> exceeds <i>Regeneration capacity (t0 < t < t1)</i>, the cell undergoes damage. Cellular damage is represented by the <i>Damage</i> variable (red curve), which has nonpositive values proportional to the light red shaded area. When <i>Chemical load</i> is below <i>Regeneration capacity</i> and <i>Fitness</i> is below of its maximal value (<i>t1 < t < t2</i>), the cell undergoes regeneration. Regeneration is represented by the <i>Regeneration</i> variable (green curve), which has nonnegative values proportional to the green shaded area. When <i>Chemical load</i> is below <i>Regeneration capacity</i> but <i>Fitness</i> is maximal (<i>t2 < t</i>), nor damage, neither regeneration occurs. <i>Fitness</i> (blue curve) is calculated by adding the (scaled) nonnegative values of <i>Regeneration</i> and the (scaled) nonpositive values of <i>Damage</i> to the maximal value of <i>Fitness</i>. See text for details.</p

PhenGreen diacetate based assay for functional studies of multidrug resistance ABC transporters.

<p>The non-fluorescent, hydrophobic PGD rapidly enters the cells through the plasma membrane. In the cytoplasm, PGD is cleaved by nonspecific esterases to yield fluorescent Phengreen (*PG—for the structures of PGD and PG see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190629#pone.0190629.s008" target="_blank">S8 Fig</a></b> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190629#pone.0190629.ref019" target="_blank">19</a>]). The ABC transporters ABCG2, ABCB1, or ABCC1 efficiently extrude PGD (and potentially also PG) to the extracellular space.</p