Molecular Dissection of Dual Pseudosymmetric Solute Translocation Pathways in Human P-Glycoprotein

Abstract

The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by highresolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.

Global alteration of the drug-binding pocket of human P-glycoprotein (ABCB1) by substitution of fifteen conserved residues reveals a negative correlation between substrate size and transport efficiency

“…Co-crystal structure of mouse P-gp with several QZ-59 cyclopeptide inhibitor derivatives [21], and PBDE-100 pesticide [31] allowed identification of several key amino acids in the substrate or modulator binding pocket of mouse P-gp. We further used published bioinformatic and mutagenesis studies, and generated a list of key amino acids that interact with different substrates or modulators [11, 17, 20, 32–34]. Additionally, we generated a homology model of human P-gp based on the refined mouse P-gp crystal structure [10] and further selected amino acids based on their position and orientation with respect to the drug-binding pocket.…”

Determining P-glycoprotein–drug interactions: Evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers

“…How interactions between drugs at the level of transport are related to the structure of the Pgp binding pocket in molecular terms is not well understood. However, the location of two pseudo-symmetric translocation pathways through the protein has recently been reported (Parveen et al, 2011). The complication of multiple binding sites and the interactions between them has not really been addressed in cell-based assays for Pgp substrates and inhibitors (Zolnerciks, Booth-Genthe, Gupta, Harris, & Unadkat, 2011), and may require the use of several reference compounds, which is not practical where many drugs are to be tested.…”

Complex Interplay between the P-Glycoprotein Multidrug Efflux Pump and the Membrane: Its Role in Modulating Protein Function

“…(32, 33, 75, 76)]. Mutagenesis studies suggested that the pocket was located at the interface between the two TM halves (77), and this was later confirmed by photoaffinity labeling in conjunction with MS (78, 79). The first X-ray crystal structures of mouse Pgp in an inward-facing conformation appeared in 2009 (34).…”