Theoretical insights on helix repacking as the origin of P-glycoprotein promiscuity

Abstract

P-glycoprotein (P-gp, ABCB1) overexpression is, currently, one of the most important multidrug resistance (MDR) mechanisms in tumor cells. Thus, modulating drug efflux by P-gp has become one of the most promising approaches to overcome MDR in cancer. Yet, more insights on the molecular basis of drug specificity and efflux-related signal transmission mechanism between the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) are needed to develop molecules with higher selectivity and efficacy. Starting from a murine P-gp crystallographic structure at the inwardfacing conformation (PDB ID: 4Q9H), we evaluated the structural quality of the herein generated human p-gp homology model. this initial human p-gp model, in the presence of the "linker" and inserted in a suitable lipid bilayer, was refined through molecular dynamics simulations and thoroughly validated. The best human P-gp model was further used to study the effect of four single-point mutations located at the TMDs, experimentally related with changes in substrate specificity and drug-stimulated ATPase activity. Remarkably, each P-gp mutation is able to induce transmembrane α-helices (TMHs) repacking, affecting the drug-binding pocket volume and the drug-binding sites properties (e.g. volume, shape and polarity) finally compromising drug binding at the substrate binding sites. Furthermore, intracellular coupling helices (ICH) also play an important role since changes in the TMHs rearrangement are shown to have an impact in residue interactions at the ICH-NBD interfaces, suggesting that identified TMHs repacking affect TMD-NBD contacts and interfere with signal transmission from the tMDs to the nBDs. Multidrug resistance (MDR) to anticancer drugs is, at the moment, a major contributor to chemotherapy failure 1. In cancer, one of the most significant MDR mechanisms results from the overexpression of P-glycoprotein, a membrane efflux pump (P-gp, ABCB1) 2. Thus, a deeper understanding on P-gp substrate specificity and efflux-related signal transmission mechanism remains crucial for the development of more potent and selective compounds able to modulate drug efflux 3. P-glycoprotein exports a broad range of structurally unrelated compounds through an ATP-dependent mechanism 4. P-gp is organized in two homologous functional units (N-and C-terminal halves) with a pseudo-2-fold symmetry. Each halve comprises one transmembrane domain (TMD), formed by six transmembrane α-helices (TMHs), and one cytoplasmic nucleotide-binding domain (NBD). Both N-and C-terminal halves are connected by a small peptide sequence (the "linker"; residues 627-688) 5,6. The TMHs are directly linked to the respective NBD by the intracellular loops, through the functional TM helices 6 (NBD1) and 12 (NBD2) and non-covalently by short intracellular coupling helices (ICHs), located between the structural TMHs 2/3 (ICH1-NBD1), 4/5 (ICH2-NBD2), 8/9 (ICH3-NBD2) and 10/11 (ICH4-NBD1) 7,8 (Fig. 1). These ICHs were found to be important for the maturation and folding of the P-gp transpo...

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

“…Each NBD/ICH pair generates a cooperative unfolding unit, 35 indicating that ICHs are important sources of communication between the TMHs and NBDs. 36 Our HDX-MS results for the ICH3 and ICH4 are consistent with this model, inasmuch as the dynamic changes with cholesterol are conserved within NBD1/ICH4 and NBD2/ICH3 pairs.…”

Effect of the Force Field on Molecular Dynamics Simulations of the Multidrug Efflux Protein P-Glycoprotein

“…The technical possibilities of molecular dynamics simulations mean that MD simulations of P-gp have been conducted with a variety of simulation conditions. To date, independent simulation studies of P-gp structures have used a range of different force fields, including several from the AMBER, ,,− CHARMM, ,− GROMOS, ,,,,,− OPLS, , and MARTINI , families of force fields. In many cases, the choice of force field is governed by the familiarity of the researcher with the particular force field or its associated MD package.…”

Lysine 268 adjacent to transmembrane helix 5 of hamster P‐glycoprotein is the major photobinding site of iodomycin in CHO B30 cells

“…Localization of the binding sites of taxol [8] (gray arrow) and iodomycin (blue arrow, this work) in the IF of human Pgp [33]. The two TMDs (TMD1: orange and TMD2: green) are linked to the NBDs NBD1 and NBD2 by coils and intracellular coupling helices (ICH1 (purple)/ICH4 (blue) with NBD1 and ICH2 (silver)/ICH3 (brown) with NBD2).…”

“…The drug-binding pocket for taxol was resolved by Cryo-EM[8]. The structural representation of the human Pgp in an IF is republished from the article by Bonito et al[33]. This open-access article is licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/ licenses/by/4.0/).…”