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. 2021 Apr 9;12(18):6293-6306.
doi: 10.1039/d0sc06288j.

Active participation of membrane lipids in inhibition of multidrug transporter P-glycoprotein

Karan Kapoor  1 , Shashank Pant  1 , Emad Tajkhorshid  1
Affiliations

Active participation of membrane lipids in inhibition of multidrug transporter P-glycoprotein

Karan Kapoor et al. Chem Sci. .

Abstract

P-glycoprotein (Pgp) is a major efflux pump in humans, overexpressed in a variety of cancers and associated with the development of multi-drug resistance. Allosteric modulation by various ligands (e.g., transport substrates, inhibitors, and ATP) has been biochemically shown to directly influence structural dynamics, and thereby, the function of Pgp. However, the molecular details of such effects, particularly with respect to the role and involvement of the surrounding lipids, are not well established. Here, we employ all-atom molecular dynamics (MD) simulations to study the conformational landscape of Pgp in the presence of a high-affinity, third-generation inhibitor, tariquidar, in comparison to the nucleotide-free (APO) and the ATP-bound states, in order to characterize the mechanical effects of the inhibitor that might be of relevance to its blocking mechanism of Pgp. Simulations in a multi-component lipid bilayer show a dynamic equilibrium between open(er) and more closed inward-facing (IF) conformations in the APO state, with binding of ATP shifting the equilibrium towards conformations more prone to ATP hydrolysis and subsequent events in the transport cycle. In the presence of the inhibitor bound to the drug-binding pocket within the transmembrane domain (TMD), Pgp samples more open IF conformations, and the nucleotide binding domains (NBDs) become highly dynamic. Interestingly, and reproduced in multiple independent simulations, the inhibitor is observed to facilitate recruitment of lipid molecules into the Pgp lumen through the two proposed drug-entry portals, where the lipid head groups from the cytoplasmic leaflet penetrate into and, in some cases, translocate inside the TMD, while the lipid tails remain extended into the bulk lipid environment. These "wedge" lipids likely enhance the inhibitor-induced conformational restriction of the TMD leading to the differential modulation of coupling pathways observed with the NBDs downstream. We suggest a novel inhibitory mechanism for tariquidar, and potentially for related third-generation Pgp inhibitors, where lipids are seen to enhance the inhibitory role in the catalytic cycle of membrane transporters.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Fig. 1
Fig. 1. Simulation systems: APO, ATP/Mg2+-bound (ATP), and TAR-bound (TAR) IF conformations of Pgp used in the simulations, with the two TMD leaflets, each connected to an NBD, colored in red and blue, respectively (see Methods for details on system construction). All the bound states were inserted in lipid bilayers containing: POPC = 40%, POPE = 43%, PYPG = 14%, and PMCL = 3% lipids matching experimental conditions in recent FRET and DEER studies on Pgp reconstituted in nanodiscs, shown in different colors. See text for chemical details of the tails.
Fig. 2
Fig. 2. Conformational dynamics of Pgp in different bound states: (A) schematic representation of Pgp depicting the geometric parameters used for monitoring its global conformational dynamics. The two halves of the protein are colored in red and blue. Conformational dynamics in all the simulated systems were evaluated by analyzing: (B) dNBD: the center of mass distance between the two NBDs, (C) γ: relative twist of the two NBDs, (D) dcyt: the distance between Cα atoms of K145 and M787 on the cytoplasmic side of the TMDs, (E) α: the angle describing relative orientation of the TMD helices measuring the degree of cytoplasmic opening, and, (F) dext: the distance between Cα atoms of S92 and R745 on the extracellular end of the TMDs. The histograms in (B–F) are calculated using the entire data set and indicate the presence of more widely open IF conformations in the TAR-bound system (shown in blue), compared to APO (green) and ATP (orange) systems. Dotted vertical lines in each panel mark the values in the recent structures for an inhibitor (zosuquidar)-bound Pgp (PDB: 6QEE) (black) and for APO Pgp (PDB: 4M1M) (purple). Time series of the distances shown in the histograms are provided in Fig. S1. (G) Representative snapshots for ATP, TARclosed, TARwide, and APO are shown.
Fig. 3
Fig. 3. Differences in secondary structure: (A) comparison of the degree of helicity in the portal helix TM10 (residues 876–889) in APO, ATP, and TAR Pgp with the zosuquidar-bound (ZOS) Pgp cryoEM structure. The analysis was performed combining all the simulations for each state. The structure of TM10 in a representative trajectory snapshot from: (B) TAR-bound (orange), (C) ATP (yellow), and, (D) APO Pgp (red) aligned with zosuquidar-bound Pgp (ZOS) (blue). Helicity of other portal helices (TM4, TM6, and TM12) is shown in Fig. S5. A p value of less than 0.001 was obtained on comparing the degree of helicity in the different systems using one-way ANOVA test, pointing to statistical significance of the results.
Fig. 4
Fig. 4. Lipid packing around and inside Pgp: the cytoplasmic-leaflet lipid density profile for (A) TAR, (B) APO, and (C) ATP Pgp simulations was calculated over all the simulated replicas for each system. The molecular systems are aligned along the Z-axis and viewed from the extracellular side of the protein, with the XY indicating the plane of the membrane. All histograms are normalized the same way, that is, with respect to the average bulk density of lipids. The lipid molecules access the central cavity through the entry portals located on either side of the TMD. TAR-bound and APO systems show enhanced lipid densities inside the central cavity of Pgp, while a low density was observed in the case of ATP-bound systems. In each panel, a part of Pgp spanning the cytoplasmic membrane leaflet (taken at the end of one of the 500 ns simulations for each system) is shown in cartoon representation with the two Pgp halves colored in green and blue, respectively.
Fig. 5
Fig. 5. Lipid access into the lumen of Pgp: (A) a lipid access event was counted if the entire head-group phosphate of any lipid accessed the central cavity of Pgp, defined by a cylinder (shown in blue) with a cross-sectional area of 300 Å2 and a height of 25 Å (see Methods for the criteria used to define the boundaries of the central cavity). Pgp is shown in a translucent surface representation. (B) Count of lipids accessing the central cavity of Pgp through the entry portals in APO (green), ATP (orange), and TAR (red) simulations, calculated by counting the number of phospholipid head groups accessing the central cavity over the course of the 500 ns simulations. Two TAR-bound conformations (TARwide and TARclosed) are defined based on the dNBD separation (Fig. 2B). In comparison to the APO and ATP systems, more lipid access events are observed in the TAR systems, especially for the TARwide conformations. A p value of less than 0.001 was obtained on comparing the lipid access events for the different systems using one-way ANOVA test, pointing to statistical significance of the results.
Fig. 6
Fig. 6. Trans-leaflet lipid translocation inside the lumen of Pgp: (A) translocation of lipids in the central cavity of Pgp that might result in their flipping was monitored by calculating the z component of the center of mass of the lipid head groups in all the simulated systems. In total, we captured 12 lipid molecules from the cytoplasmic (lower) leaflet that undergo partial flipping during the simulations. Data for the APO Pgp are shown in Fig. S7. (B–D) Lipids (licorice representation) penetrating into the central cavity of (B) TAR-bound, (C) APO and (D) ATP Pgp (translucent surface representation) from representative snapshots of the simulations. Phosphorus atoms from the lipid head groups are shown as tan beads to delineate the membrane boundaries. Inset in (B) shows the closeup view of the interactions between the bound TAR (thick sticks) and the flipping lipid (PE) molecule showing the largest translation (black trace in (A)) into the other side of the membrane, as well as the surrounding Pgp residues within 3.5 Å of either TAR or the head group of the translocating (PE) lipid. A 2D representation of the TAR binding pocket along with the flipping PE molecule is shown in Fig. S2B.
Fig. 7
Fig. 7. Allosteric coupling between TMDs and NBDs: The coupling pathway calculated for a representative 500 ns long simulation of: (A) ATP, (B) APO, and (C) TAR-bound Pgp, between S988 in TMD (the source) and Q1114 in the NBD (the sink), is shown in green. Q-loop (containing conserved Q1114) is shown in yellow, coupling helices in purple, A-loop (containing conserved Y1040) in orange, and the rest of the protein in a transparent cartoon representation. All the residues that form the allosteric pathway are highlighted in the representative replica for each system. In the majority of TAR-bound simulations, extended coupling pathways passing primarily through the coupling helices and the Q-loop in the NBDs are observed. Shorter and more direct pathways connecting the TMD and the NBD are observed in the ATP and APO simulations.
Fig. 8
Fig. 8. Modulation of the conformational dynamics of Pgp by the lipidic environment and bound inhibitor: In nucleotide-free (APO) state, Pgp exists in a dynamic equilibrium between open and closed IF conformations. ATP binding in the NBDs results in shifting of the equilibrium towards predominantly closed IF conformations of Pgp. The presence of a third-generation inhibitor (TAR) leads to increased lipid recruitment into the central cavity of Pgp, thereby, imposing a stronger inhibitory effect on NBD dimerization and closure of the cytoplasmic side of the TMDs.
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