Energy transduction and alternating access of the mammalian ABC transporter P-glycoprotein

Verhalen, Brandy; Dastvan, Reza; Thangapandian, Sundarapandian; Peskova, Yelena; Koteiche, Hanane A.; Nakamoto, Robert K.; Tajkhorshid, Emad; Mchaourab, Hassane S.

doi:10.1038/nature21414

Cited by 202 publications

(247 citation statements)

References 46 publications

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“…The resolution of those structures, however, does not allow for detailed analysis of the solvent accessibility of the NBD-NBD interface. In contrast, the elegant DEER studies by Verhalen et al (30) indicate that most of the population shifts in the presence of verapamil, vanadate, and ATP so that the NBDs are in close proximity, but residual components of the population with partially separated NBDs remain. Those studies were also done in micelles, and did not include the state analogous to our vanadate trapped state.…”

Section: Discussionmentioning

confidence: 57%

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale

Guttman

Atkins

2018

Journal of Biological Chemistry

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P-glycoprotein (P-gp) is a highly substrate promiscuous efflux transporter that plays a critical role in drug disposition. P-gp utilizes ATP hydrolysis by nucleotide binding domains (NBDs) to drive transitions between inwardfacing (IF) conformations that bind drugs and outward-facing (OF) conformations that release them to the extracellular solution. However, the details of the protein dynamics within either macroscopic IF or OF conformation remain uncharacterized, and the functional role of local dynamics has not been determined. In this work we measured the local dynamics of the IF state of P-gp in lipid nanodiscs and in detergent solution by H/D exchange mass spectrometry. We observed 'EX1 exchange kinetics,' or bimodal kinetics, for several peptides distributed in both NBDs, particularly for P-gp in the lipid nanodiscs. Remarkably, the EX1 kinetics occurred on several time scales, ranging from seconds to hours, suggesting highly complex, and correlated, motions. The results indicate at least three distinct conformational states in the ligand free P-gp and suggest a rough conformational landscape. Addition of excess ATP and vanadate, to favor the OF conformations, caused a generalized, but modest, decrease in H/D exchange throughout the NBDs and slowed the EX1 kinetic transitions of several peptides. The functional implications of the results are consistent with the possibility that conformational selection provides as a source of substrate promiscuity.The membrane transporter P-glycoprotein plays a critical role in disposition and distribution for a wide range of therapeutic drug classes and it is a major determinant of cancer cell drug resistance (1-3). Among the complex properties of P-gp, the mechanisms by which it achieves its extraordinary substrate promiscuity have not been fully understood. P-glycoprotein is a member of the ABC transporter family (ATP Binding Cassette), which includes many structurally related transporters that utilize ATP hydrolysis to transport lipophilic substrates. P-gp includes 12 transmembrane helices (TMHs) that comprise the drug binding site and two NBDs that alternately bind and hydrolyze ATP (4-6). The linear sequence of P-gp includes an N-terminal transmembrane domain containing six TMHs collectively referred to as "transmembrane domain 1" (TMD1), followed by NBD1, a linker region, six additional TMHs, collectively called "transmembrane domain 2" (TMD2), and a second NBD (NBD2). The three dimensional structures of murine and C. elegans orthologs have provided a valuable model for the human Pgp, for which no crystal structure is available ( Figure 1-mouse P-gp structures). The available crystal structures from multiple homologous ABC transporters, including Sav1866 and MsbA (7-9), combined with cryo EM structures, DEER studies with spin-labeled variants, and FRET with fluorescently-labeled NBDs, suggest that P-gp undergoes large scale opening and closing motions, between an inward facing (IF) conformational ensemble and outward-facing (OF) conformational ensemble (1...

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Section: Discussionmentioning

confidence: 57%

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale

Guttman

Atkins

2018

Journal of Biological Chemistry

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“…These data also suggest that the transition between these conformations requires ATP hydrolysis [8, 12]. It is proposed that binding of amphipathic agents to the drug-binding pocket and ATP hydrolysis results in open to closed conformational switch and release of substrate into the extracellular space [13, 14]. …”

Section: Introductionmentioning

confidence: 99%

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

Vahedi

Chufán

Ambudkar

2017

Biochemical Pharmacology

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P-glycoprotein (P-gp), an ATP-dependent efflux pump, is linked to the development of multidrug resistance in cancer cells. However, the drug-binding sites and translocation pathways of this transporter are not yet well-characterized. We recently demonstrated the important role of tyrosine residues in regulating P-gp ATP hydrolysis via hydrogen bond formations with high affinity modulators. Since tyrosine is both a hydrogen bond donor and acceptor, and non-covalent interactions are key in drug transport, in this study we investigated the global effect of enrichment of tyrosine residues in the drug-binding pocket on the drug binding and transport of P-gp. By employing computational analysis, 15 conserved residues in the drug-binding pocket of human P-gp that interact with substrates were identified and then substituted with tyrosine, including 11 phenylalanine (F72, F303, F314, F336, F732, F759, F770, F938, F942, F983, F994), two leucine (L339, L975), one isoleucine (I306), and one methionine (M949). Characterization of the tyrosine-rich P-gp mutant in HeLa cells demonstrated that this major alteration in the drug-binding pocket by introducing fifteen additional tyrosine residues is well tolerated and has no measurable effect on total or cell surface expression of this mutant. Although the tyrosine-enriched mutant P-gp could transport small to moderate size (<1000 Daltons) fluorescent substrates, its ability to transport large (>1000 Daltons) substrates such as NBD-cyclosporine A, Bodipy-paclitaxel and Bodipy-vinblastine was significantly decreased. This was further supported by the physico-chemical characterization of seventeen tested substrates, which revealed a negative correlation between drug transport and molecular size for the tyrosine-enriched P-gp mutant.

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“…Based on the Anfinsen dogma, it became customary to explain function in terms of a single conformation or of well-defined transitions between a few conformations defined at atomic resolution. While this is certainly a reasonable approximation in some cases [75][76][77], availability of distance distributions demonstrates that rather often conformation transitions are coupled to order-disorder transitions or are shifts in disorder equilibria [39,[78][79][80][81][82][83][84][85][86][87][88][89][90][91][92][93][94]. Among the systems addressed by PDS to date, the fraction where at least one state is genuinely disordered is surprisingly large.…”

Section: A Fuzzy Relation Of Structure To Functionmentioning

confidence: 99%

The contribution of modern EPR to structural biology

Jeschke

2018

Emerging Topics in Life Sciences

126

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Electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labelling is applicable to biomolecules and their complexes irrespective of system size and in a broad range of environments. Neither short-range nor long-range order is required to obtain structural restraints on accessibility of sites to water or oxygen, on secondary structure, and on distances between sites. Many of the experiments characterize a static ensemble obtained by shock-freezing. Compared with characterizing the dynamic ensemble at ambient temperature, analysis is simplified and information loss due to overlapping timescales of measurement and system dynamics is avoided. The necessity for labelling leads to sparse restraint sets that require integration with data from other methodologies for building models. The double electron-electron resonance experiment provides distance distributions in the nanometre range that carry information not only on the mean conformation but also on the width of the native ensemble. The distribution widths are often inconsistent with Anfinsen's concept that a sequence encodes a single native conformation defined at atomic resolution under physiological conditions.

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Energy transduction and alternating access of the mammalian ABC transporter P-glycoprotein

Cited by 202 publications

References 46 publications

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale

Conformational dynamics of P-glycoprotein in lipid nanodiscs and detergent micelles reveal complex motions on a wide time scale

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

The contribution of modern EPR to structural biology

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