Nucleotide-induced Structural Changes in P-glycoprotein Observed by Electron Microscopy

Lee, Jyh-Yeuan; Urbatsch, Ina L.; Senior, Alan E.; Wilkens, Stephan

doi:10.1074/jbc.m707028200

Cited by 67 publications

(56 citation statements)

References 48 publications

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“…The conformational analysis is strikingly analogous to the results based on two-dimensional crystal analysis wherein the low resolution electron density suggests a difference in the accessibility to solvent of the drug-binding site in the trapped state, which is inaccessible, as compared with the ATP analogbound and ADP-bound states, for which the channel is more open (46). The epitopes for UIC2 and MRK16 are both discontinuous and made up of multiple extracellular loops (Fig.…”

Section: Discussionmentioning

confidence: 55%

Conformational Analysis of Human ATP-binding Cassette Transporter ABCB1 in Lipid Nanodiscs and Inhibition by the Antibodies MRK16 and UIC2

Ritchie

Kwon

Atkins

2011

Journal of Biological Chemistry

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Background: P-glycoprotein plays a role in drug resistance and drug interactions. Results: Surface plasmon resonance with P-glycoprotein in lipid nanodiscs indicates that inhibitory antibodies uncouple the ATP hydrolysis of P-gp from its transport function. Conclusion: Inhibitory antibodies do not prevent P-gp flux through the catalytic cycle, but rather block drug efflux without inhibiting ATP hydrolysis. Significance: The results clarify the mechanism of inhibitory antibodies to P-gp.

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Section: Discussionmentioning

confidence: 55%

Conformational Analysis of Human ATP-binding Cassette Transporter ABCB1 in Lipid Nanodiscs and Inhibition by the Antibodies MRK16 and UIC2

Ritchie

Kwon

Atkins

2011

Journal of Biological Chemistry

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“…The available information from electron microscopy of ABC transporters in a lipid membrane corresponds to snapshots at low resolution that do not provide a unified and definitive picture. For example, a study of BmrA suggests large conformational changes during the hydrolysis cycle (41), whereas a study of P-glycoprotein suggests small conformational changes elicited by substrates and nucleotides (42). In general, available studies of BmrA and MsbA suggest similarity between the conformational changes of these proteins in detergent and liposomes (23,40,43).…”

Section: Discussionmentioning

confidence: 99%

The Lipid Bilayer Modulates the Structure and Function of an ATP-binding Cassette Exporter

Zoghbi

Cooper

Altenberg

2016

Journal of Biological Chemistry

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ATP-binding cassette exporters use the energy of ATP hydrolysis to transport substrates across membranes by switching between inward-and outward-facing conformations. Essentially all structural studies of these proteins have been performed with the proteins in detergent micelles, locked in specific conformations and/or at low temperature. Here, we used luminescence resonance energy transfer spectroscopy to study the prototypical ATP-binding cassette exporter MsbA reconstituted in nanodiscs at 37°C while it performs ATP hydrolysis. We found major differences when comparing MsbA in these native-like conditions with double electron-electron resonance data and the crystal structure of MsbA in the open inward-facing conformation. The most striking differences include a significantly smaller separation between the nucleotide-binding domains and a larger fraction of molecules with associated nucleotide-binding domains in the nucleotide-free apo state. These studies stress the importance of studying membrane proteins in an environment that approaches physiological conditions. ATP-binding cassette (ABC)2 transporters constitute one of the largest families of membrane proteins and are found in all domains of life (1-3). They can be either importers (most prokaryote ABC transporters) or exporters (most mammal ABC transporters) (1-3). The core structure of ABC proteins consists of two transmembrane domains that form the translocation pathway and two conserved nucleotide-binding domains (NBDs) that bind and hydrolyze ATP (1-3), a process essential for their function. Binding of ATP promotes the formation of an NBD dimer in a head to tail orientation (4, 5). In the resulting structure two ATP molecules are "sandwiched" at the dimer interface, and residues from both NBDs form each of the two nucleotide-binding sites (1, 4, 5). NBD dimerization is essential for ATP hydrolysis and requires ATP binding to both nucleotide-binding sites (6), whereas dissociation of the dimers occurs following hydrolysis at only one of the two sites (7). This ATP-dependent NBDs dimerization/dissociation process is coupled to rearrangements of the transmembrane helices, switching the transporters from an inward-facing conformation (dissociated NBDs) to an outward-facing conformation (dimeric NBDs), with the concomitant translocation of substrate (1, 3).Two of the most studied ABC exporters are the multidrug resistance protein P-glycoprotein (MDR1 and ABCB1) that plays a role in the resistance to chemotherapy of some forms of cancer (3),and its bacterial homolog, the lipid flippase MsbA (8). MsbA is located in the inner membrane of Gram-negative bacteria, where it transports lipid A from the inner to the outer leaflet (9, 10). MsbA has been crystallized in inward-and outward-facing conformations (11) and has also been extensively studied by different spectroscopic techniques (12-18). Several studies point to large motions between the nucleotide-free "open" inward-facing conformation (NBDs separated by tens of Angstroms) and the ATP-bound "closed" outw...

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“…[16][17][18][19] High-resolution structures and NMR, EPR, and electron microscopy data strongly suggest that ABC exporters undergo large movements during substrate efflux, in particular upon ATP binding and/or hydrolysis. [20][21][22][23][24][25][26][27] Although structure determinations and the large body of experimental data obtained on ABC transporters have shed light on the transport mechanism, the answers to some questions remain elusive: what are the conformational changes occurring upon substrate binding and ATP binding and/or hydrolysis, and how do these changes relate to solute translocation? Molecular dynamics (MD) simulations are a means of exploring the conformational dynamics of proteins with an extreme level of detail as regards atomic motions.…”

Section: Introductionmentioning

confidence: 99%

Dynamics and Structural Changes Induced by ATP Binding in SAV1866, a Bacterial ABC Exporter

et al. 2010

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Multidrug transporters of the ATP-binding cassette family export a wide variety of compounds across membranes in both prokaryotes and eukaryotes, using ATP hydrolysis as energy source. Several of these membrane proteins are of clinical importance. Although biochemical and structural studies have provided insights into the mechanism underlying substrate transport, many key questions subsist regarding the molecular and structural nature of this mechanism. In particular, the detailed conformational changes occurring during the catalytic cycle are still elusive. We explored the conformational changes occurring upon ATP/Mg 2+ binding using molecular dynamics simulations starting from the nucleotide-bound structure of SAV1866 embedded in an explicit lipid bilayer. The removal of nucleotide revealed a major rearrangement in the outer membrane leaflet portion of the transmembrane domain (TMD) resulting in the closure of the central cavity at the extracellular side. This closure is similar to that observed in the crystal nucleotide-free structures. The interface of the nucleotide-binding domain dimer (NDB) is significantly more hydrated in the nucleotide-free trajectory though it is not disrupted. This finding suggests that the TMD closure could occur as a first step preceding the dissociation of the dimer. The transmission pathway of the signal triggered by the removal of ATP/Mg 2+ mainly involves the conserved Q-loop and X-loop as well as TM6.

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Nucleotide-induced Structural Changes in P-glycoprotein Observed by Electron Microscopy

Cited by 67 publications

References 48 publications

Conformational Analysis of Human ATP-binding Cassette Transporter ABCB1 in Lipid Nanodiscs and Inhibition by the Antibodies MRK16 and UIC2

Conformational Analysis of Human ATP-binding Cassette Transporter ABCB1 in Lipid Nanodiscs and Inhibition by the Antibodies MRK16 and UIC2

The Lipid Bilayer Modulates the Structure and Function of an ATP-binding Cassette Exporter

Dynamics and Structural Changes Induced by ATP Binding in SAV1866, a Bacterial ABC Exporter

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