Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9 Å resolution

Thonghin, Nopnithi; Collins, Richard F.; Barbieri, Alessandro; Shafi, Talha; Siebert, Alistair; Ford, Robert C.

doi:10.1186/s12900-018-0098-z

Cited by 32 publications

(43 citation statements)

References 49 publications

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“…In order to understand the nature of the TMH4 and TMH10 alterations in the human P-gp cryo-EM structure, four mouse P-gp crystallographic structures in the apo state inward-facing conformation (PDB IDs: 4M1M, 4Q9H, 5KPI 111 and 6GDI 112 ) and in the presence of ligands at the DBP (PDB IDs: 4M2S 113 and 4Q9L 61 ) were used for comparison. Visual inspection showed that both TMH4 and 10 are continuous α-helices in all apo and holo P-gp crystallographic structures considered, as observed in our human WT P-gp model.…”

Section: Resultsmentioning

confidence: 99%

“…Herein, while both H and R sites were initially characterized by their interaction with Hoechst 33342 108 or Rhodamine-123 35 , respectively, the modulator M site was identified from the localization of the co-crystallized ligands QZ-SSS and QZ-RRR in the first crystallographic structure of murine P-gp 5 . Regarding SBSs, both were later characterized by molecular docking 40 and experimentally confirmed by electron microscopy 112 .…”

Section: Structural Analysis Of the Human P-gp Variants To Gain Addimentioning

confidence: 99%

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

Bonito

Ferreira

et al. 2020

Sci Rep

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Structural Analysis Of the Human P-gp Variants To Gain Addimentioning

confidence: 99%

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

Bonito

Ferreira

et al. 2020

Sci Rep

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“…Structures for four of the five nonsymmetric human transporters from the ABCB subfamily have been solved (ABCB1, ABCB2/B3, ABCB4, ABCB11). Human ABCB1 [16,54,55] and homologues (mouse [56,57] Caenorhabditis elegans [58] C. merolae [59,60]) are full-length transporters, in which the sequences of the N and the C-halves have diverged from an initial putative gene duplication, maintaining pseudo twofold symmetry, but the full-length transporter can also be interpreted as a heterodimer with two canonical NBSs. The conformations of the outward-facing and the occluded state are largely symmetric showing pseudo twofold symmetry, while Fig.…”

Section: Tmrabmentioning

confidence: 99%

The role of the degenerate nucleotide binding site in type I ABC exporters

2020

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ATP-binding cassette (ABC) transporters are fascinating molecular machines that are capable of transporting a large variety of chemically diverse compounds. The energy required for translocation is derived from binding and hydrolysis of ATP. All ABC transporters share a basic architecture and are composed of two transmembrane domains and two nucleotide binding domains (NBDs). The latter harbor all conserved sequence motifs that hallmark the ABC transporter superfamily. The NBDs form the nucleotide binding sites (NBSs) in their interface. Transporters with two active NBSs are called canonical transporters, while ABC exporters from eukaryotic organisms, including humans, frequently have a degenerate NBS1 containing noncanonical residues that strongly impair ATP hydrolysis. Here, we summarize current knowledge on degenerate ABC transporters. By integrating structural information with biophysical and biochemical evidence of asymmetric function, we develop a model for the transport cycle of degenerate ABC transporters. We will elaborate on the unclear functional advantages of a degenerate NBS.

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“…Pgp undergoes dynamic conformational changes to allow for an array of substrate binding and efflux which is associated with ATP binding and hydrolysis on the cytoplasmic side allowing unidirectional outward flow of the substrates. Thermodynamic studies have demonstrated that while inward V-conformation is energetically-feasible conformation and transient outward facing conformation is adopted at high-energy state with the consumption of ATP-derived energy (46). In spite of the controversies in the reported crystal structure regarding the location of ATP-binding domain due to the use of detergents, and the absence of nucleotides to obtain the crystal structure, however, it is well-documented that the two ATP-binding domains should be on the intracellular side as the cellular concentration of ATP (1-10 mM) far exceeds the domain binding constant (∼0.01 mM) (47,48).…”

Section: P-gp: Protein Expression Structure and Functionmentioning

confidence: 99%

Perplexing Role of P-Glycoprotein in Tumor Microenvironment

2020

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Development of multidrug resistance (MDR) still remains a major obstacle to the long-term success of cancer therapy. P-glycoprotein (P-gp) is a well-identified membrane transporter with capability to efflux drug molecules out of the cancer cell leading to reduced efficiency of chemotherapy. Cancer cells upregulate P-gp expression as an adaptive response to evade chemotherapy mediated cell death. While several P-gp inhibitors have been discovered by in silico and pre-clinical studies, very few have successfully passed all phases of the clinical trials. Studies show that application of P-gp inhibitors in cancer therapy regimen following development of MDR achieved limited beneficial outcomes. While, the non-specific substrate binding to P-gp has made the drug-design a challenge, a bigger perplexing challenge comes from its role in tumor immunology. Expression of P-gp was noted immune cell phenotypes with apparently antagonistic functionality. Both pro-tumor M 2-macrophages and, anti-tumor NK-cell and Th17/CD4 + T cell subsets have shown enhanced expression of P-gp. While drug based inhibition of P-gp in pro-tumor immune cell phenotypes could promote tumor elimination, however, it would not be a rational choice to exert inhibition of P-gp on anti-tumor immune cell phenotypes. This mutually exclusive paradigm of P-gp functionality requires a more comprehensive and detailed understanding of its role in tumor microenvironment with active interplay of cancer and immune cells in the tumor mileu. In this review, we focus on the current understanding of the role of P-gp in cancer cells and immune cells and finally attempt to highlight some caveats in the current understanding of its role in comprehensive tumor microenvironment along with challenges in the development of P-gp inhibitors toward anti-cancer therapy.

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Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9 Å resolution

Cited by 32 publications

References 49 publications

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

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

The role of the degenerate nucleotide binding site in type I ABC exporters

Perplexing Role of P-Glycoprotein in Tumor Microenvironment

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