On the Origin of Large Flexibility of P-glycoprotein in the Inward-facing State

Wen, Po‐Chao; Verhalen, Brandy; Wilkens, Stephan; Mchaourab, Hassane S.; Tajkhorshid, Emad

doi:10.1074/jbc.m113.450114

Cited by 128 publications

(164 citation statements)

References 86 publications

(90 reference statements)

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“…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" outward-facing conformation (tightly associated NBDs). These observations disagree with evidence suggesting that the NBDs remain in close contact at all times during the transport cycle and that such large conformational changes might not be physiological (19 -22).For simplicity and because of technical limitations, essentially all structural studies of ABC exporters have been performed with the proteins solubilized in detergent, locked in specific conformations and/or at low temperature (11,12,14,18,(23)(24)(25)(26). However, understanding of the structure-function of membrane proteins requires their study under more physiological conditions, at a minimum: 1) reconstitution into lipid bilayers, their native environment; 2) physiological temperatures; and 3) functioning conditions, as opposed to specific locked conformations.…”

contrasting

confidence: 44%

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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contrasting

confidence: 44%

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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“…The overall range of distances between residues 626 (C-term of NBD1) to 1271 (C-term of NBD2) sampled by the three inward-facing conformations presented here is ∼29-36 Å, whereas the corresponding distance in the original published structure (9) is only ∼13 Å. Such dynamic conformational flexibility in the inward-facing state has also been observed in biochemical, biophysical, and molecular dynamics simulations experiments on bacterial ABC transporters, such as MsbA (13) and BmrA (16), and recently on mouse P-gp (25). The enlargement of the portals (formed from TM4/TM6 and TM10/TM12) facing the inner-membrane leaflet side may be required for larger substrates like β-amyloid (∼4 kDa in size) (26) to enter and bind inside the substratebinding pocket.…”

Section: Discussionmentioning

confidence: 68%

“…The X-ray structures of P-gp described here, along with those published (9), will be useful for the molecular modeling of conformational trajectories to understand how the substratebinding pocket changes during the transport cycle (25). Because human and mouse P-gp share nearly 87% protein sequence identity, our structures present experimentally derived checkpoints useful for docking simulations.…”

Section: Discussionmentioning

confidence: 93%

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

Ward

Szewczyk²,

Grimard

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

236

224

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P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATPBinding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse Pgp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.

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“…In the case of ABCB1, Wen, Verhalen, Wilkens, McHaourab, and Tajkhorshid (2013) performed MD simulations. According to their results, the lipid enters between TM3, 4, and 6 and interacts with Leu-300, Ala-302, Tyr-303, and Ala-338, previously shown to be involved in drug binding (for details see Section 5).…”

Section: P0360mentioning

confidence: 99%

“…p0420 Wen et al (2013) have performed MD simulations using the mouse Abcb1 (P-gp) structure (PDB:3G5U) in the apo conformation, with the NBDs apart. Since the dynamics and lipid interactions in the simulations may be affected by the initial lipid contacts of the protein, they have generated four different systems with the protein differently embedded in a bilayer built from POPE, for parallel simulations.…”

Section: Article In Pressmentioning

confidence: 99%