Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers into Monomers during the Hydrolysis Cycle

Zoghbi, Maria E.; Krishnan, S.; Altenberg, Guillermo A.

doi:10.1074/jbc.m112.340281

Cited by 18 publications

(55 citation statements)

References 26 publications

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“…There is an unavoidable uncertainty about the position of the optical probes because of their length, which introduces an error when distances between the probes are assumed to be those between the reacted thiols in the Cys side chains. However, because measurements from others and us have shown remarkably close distances to those expected from crystal structures (18,28,30,36), LRET seems more than adequate to address domain movements in MsbA.…”

Section: Methodsmentioning

confidence: 67%

“…LRET Decays during the ATP Hydrolysis Cycle-LRET is a very sensitive technique to measure changes in distance between donor-acceptor probes attached to a protein (18,28,30,38). Distances can be calculated from the sensitized emission decay of the acceptor after a single excitation pulse, as described under "Experimental Procedures" and previous publications (18,28).…”

Section: Resultsmentioning

confidence: 99%

“…LRET-LRET recordings were performed essentially as described (6,7,18,28). Nanodisc samples containing 0.1-0.5 m of labeled MsbA were analyzed in 3-mm path length quartz cuvettes before and after successive additions of ATP, MgSO 4 , and sodium orthovanadate (Vi).…”

Section: Methodsmentioning

confidence: 99%

“…All Bodipy FL sensitized emission decays (recorded at 520 nm) were well fitted by a three-exponential decay. However, the fastest component (shortest distance; DA ϭ 100 -200 s) was disregarded for the calculations of distances and percentage of molecules in each conformation because the contribution of this exponential component to the whole signal was Յ5% and did not change appreciably under different experimental conditions, and it is likely the result of a very small fraction of protein aggregates and/or instrument artifacts, as described (18,28). The number of donor-acceptor pairs that contribute to a distance component (percentage MsbA molecules in each conformation) was estimated from the relative intensity of each lifetime component corrected by the rate of energy transfer (k ϭ 1/ DA Ϫ 1/ D ) (18,33).…”

Section: Methodsmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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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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“…Finally, hydrolysis of either one or both molecules of ATP disrupts the closed NBD dimer and restores the ABC transporter in its original configuration. The driving force for this reaction comes from an increase in negative charge at the NBD due to the formation of ADP and Pi with subsequent electrostatic repulsion [75].…”

Section: Putative Catalytic Mechanism Of Abc Transporters Of the "Expmentioning

confidence: 99%

Structure and mechanism of ATP-dependent phospholipid transporters

López‐Marqués

Poulsen

Bailly

et al. 2015

Biochimica et Biophysica Acta (BBA) - General Subjects

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Background: ATP-binding cassette (ABC) transporters and P4-ATPases are two large and seemingly unrelated families of primary active pumps involved in moving phospholipids from one leaflet of a biological membrane to the other. Scope of review: This review aims to identify common mechanistic features in the way phospholipid flipping is carried out by two evolutionarily unrelated families of transporters. Major conclusions: Both protein families hydrolyze ATP, although they employ different mechanisms to use it, and have a comparable size with twelve transmembrane segments in the functional unit. Further, despite differences in overall architecture, both appear to operate by an alternating access mechanism and during transport they might allow access of phospholipids to the internal part of the transmembrane domain. The latter feature is obvious for ABC transporters, but phospholipids and other hydrophobic molecules have also been found embedded in P-type ATPase crystal structures. Taken together, in two diverse groups of pumps, nature appears to have evolved quite similar ways of flipping phospholipids. General significance: Our understanding of the structural basis for phospholipid flipping is still limited but it seems plausible that a general mechanism for phospholipid flipping exists in nature. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.

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ABC Exporters in Pathogenesis: Role of Synthetic Anti-Microbial Peptides

Kabra

Singh

2020

Protein J

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Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers into Monomers during the Hydrolysis Cycle

Cited by 18 publications

References 26 publications

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

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

Structure and mechanism of ATP-dependent phospholipid transporters

ABC Exporters in Pathogenesis: Role of Synthetic Anti-Microbial Peptides

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