Cholesterol Asymmetrically Modulates the Conformational Ensemble of the Nucleotide-Binding Domains of P-Glycoprotein in Lipid Nanodiscs

Abstract

P-Glycoprotein (P-gp) is an ATP-dependent efflux pump that clears a wide variety of drugs and toxins from cells. P-gp undergoes large-scale structural changes and demonstrates conformational heterogeneity even within a single catalytic or drug-bound state, although the role of heterogeneity remains unclear. P-gp is found in a variety of cell types that vary in lipid composition, which modulates its activity. An understanding of structural or dynamic changes due to the lipid environment is lacking. We aimed to determine the effects of cholesterol in a membrane on the conformational behavior of P-gp in lipid nanodiscs. The presence of cholesterol stimulates ATP hydrolysis and alters lipid order and fluidity. Hydrogen/deuterium exchange mass spectrometry demonstrates that cholesterol in the membrane induces asymmetric, long-range changes in the distributions and exchange kinetics of conformations of the nucleotidebinding domains, correlating the effects of lipid composition on activity with specific changes in the P-gp conformational landscape.

Long Range Communication between the Drug-Binding Sites and Nucleotide Binding Domains of the Efflux Transporter ABCB1

“…In our comparison of P-gp dynamics between lipid environments (P-gp in DMPC NDs with or without cholesterol), the majority of affected peptides showed bimodal isotopic distributions. 27 We find with drug binding that most of the peptides that are more strongly affected also show bimodal distributions, consistent with a role for conformational heterogeneity in drug binding.…”

“…The observed effects due to either vinblastine or zosuquidar binding to P-gp-NDs are somewhat different from those we observed when cholesterol is included in the NDs. 27 This suggests that our previously reported effects from cholesterol are predominantly due to environmental changes in the ND as opposed to cholesterol in the binding pocket. The predominant effects with cholesterol are increased protection throughout NBD2 and ICH3 through a shift in the equilibrium distribution of conformations within the apo macrostate; modest increases in exchange are observed in NBD1/ICH4.…”

“…As previously reported, P-gp in lipid NDs and detergent/lipid micelles exhibits EX1 kinetics, or conformational exchange, across a broad range of timescales. 25 , 27 This behavior is observed via bimodal isotopic distributions in the mass spectrum for a given peptide. Specifically, one can observe two slowly exchanging states for a region if the two conformations differ in their degree of solvent exposure or hydrogen bonding, and therefore deuterium uptake.…”

“…In fact, EPR-based DEER studies, 20 , 21 LRET, 22 atomic force microscopy (AFM), 23 , 24 and H/D exchange mass spectrometry (HDX-MS) 25 − 27 have demonstrated the highly dynamic nature of P-gp, which is critical for its catalytic cycle that requires large-scale conformational changes and for its substrate promiscuity. MD simulations have also been critical for supporting and driving mechanistic studies of conformational changes in P-gp.…”

Mechanistic Insights Delineating the Role of Cholesterol in Epithelial Mesenchymal Transition and Drug Resistance in Cancer

“…Interestingly, evidence exists highlighting the role of cholesterol in altering P-gp substrate K M values thereby implying that cholesterol directly interacts with substrate binding sites (Hegedüs et al, 2015). This is illustrated mechanistically, by studies conducted by Clouser et al proposing that plasma membrane cholesterol modulates P-gptransmembrane helix-membrane interactions which consequently promotes long-range conformational changes in the nucleotide-binding domain of P-gp promoting a drastic increase in the rate of ATP hydrolysis (Clouser et al, 2021). This consequently justifies the crucial role cholesterol plays in supporting the activity of P-gp housed within lipid rafts.…”