Tracing the substrate translocation mechanism in P-glycoprotein

Abstract

P-glycoprotein (Pgp) is a highly dynamic ATP-binding cassette transporter that confers cancer multidrug resistance and mediates the bioavailability and pharmacokinetics of many drugs. Structural and biochemical data have provided insights into the binding of diverse compounds, but how they are translocated through the membrane has remained elusive. The biggest hurdle to address this fundamental question has been isolating and characterizing the short-lived transition states during transport. Here, we covalently attached a cyclopeptide substrate to discrete sites of Pgp. Cryo-EM analyses of inward- and outward-facing conformations enabled tracing of the substrate passage and revealed how a cascade of conformational changes in transmembrane helix 1 drives substrate movement across the membrane bilayer upon ATP binding. Observation of a double substrate-bound outward-facing conformation suggests that Pgp can co-transport two compounds simultaneously.One-Sentence SummaryCryo-EM reveals the substrate transport pathway and mechanism of P-glycoprotein from initial binding to release.

R6G narrows BmrA conformational spectrum for a more efficient use of ATP

“…To exemplify this, we have modelled R6G in 2 conformations of the benzoate moiety with 180° flip of the xanthene core fitting equally well (Figure 2C, left). In E504A apo , E504A apo-25µMATP and E504A apo-100µMATP , multiple IF conformations of BmrA are observed with a rearrangement of TM helices and the NBD (Figure 2A-C), in line with the flexibility observed for BmrA [16,21]and other ABC transporters in this conformation [4,7,23]. Notably, the E504A apo structure is exactly the same as the WT or A582C mutant structures recently solved (rmsd = 0.52Å over 902 atoms and 0.45 Å over 883 atoms, respectively; suppFigure 11 [21]).…”

“…These transporters thus harbor a polyspecificity for multiple ligands, that they can accommodate within a rather large and size adaptable binding pocket. The wealth of structural data available on these transporters as well as the biochemical and biophysical characterizations have highlighted their plasticity and their ability to adapt to various ligands [4][5][6][7][8][9]. At the core, it is clear that ABC transporters are very flexible and undergo significant conformational changes to perform their transport function.…”

Coupling the role of lipids to the conformational dynamics of the ABC transporter P-gp

“…This pattern was recently confirmed across species, as evidenced by the straight conformation observed in the mouse P-gp structure solved in detergent. 36 In our simulations we mimicked the highly specialized canalicular membrane composition, enriched with sphingomyelin and cholesterol, which can withstand the harsh environment formed by the bile molecular soap. 17 To better understand the dynamics and structural relationships of helices TM4 and TM10 under various conditions, the two conformers were simulated under native-like conditions and in different environments by removing cholesterol and ATP, known regulators of P-gp dynamics, individually and in combination, to explore their effects on the structural dynamics and conformational ensembles.…”

“…57,58 For P-pg, computational investigations have provided insights into the conformational flexibility of the membrane-embedded transporter, offering valuable insights into its structural dynamics, accessibility of its transmembrane cavity to lipids and other membrane components, and substrate binding. 11,12,27,36,[59][60][61][62][63][64][65][66] Here we employ MD simulations to investigate P-gp structural dynamics in asymmetric multi-component bilayers that mimic the canalicular membrane of hepatocytes. The structural determinants governing the two distinct P-gp inward-open conformations, straight and kinked, are compared.…”

“…From a structural biology perspective, the community has made tremendous progress, through the determination of numerous high-resolution P-gp structures in both inwardopen [35][36][37][38][39][40][41][42][43] and outward-open conformations. 36,43,44 The majority of these structures have been derived from mouse and other organisms, 43,45 employing lipid-detergent mixtures that, although providing an environment that is a quite different from a cellular membrane, can provide a valuable means to study ABC transporters in vitro and in vivo. 46,47 The recent advances in cryo-electron microscopy (cryo-EM) combined with nanodisc technology, have facilitated the acquisition of high-resolution structural data of P-gp in a defined lipid environment.…”