A Tweezers-like Motion of the ATP-Binding Cassette Dimer in an ABC Transport Cycle

Chen, Jue; Lü, Gang; Lin, Jeffrey S.; Davidson, Amy L.; Quiocho, Florante A.

doi:10.1016/j.molcel.2003.08.004

Cited by 471 publications

(679 citation statements)

References 47 publications

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“…Our DLS and ITC experiments indicate, however, that the dimer contains two nucleotides, confirming previous structural information about NBD dimers (6,(8)(9)(10). Moreover, previous size-exclusion experiments with E166Q GlcV (23) and analysis of the nucleotide composition of three trapped intermediate states of the NBD of Mdl1p, a mitochondrial peptide ABC transporter (15), also indicated a stoichiometry of two nucleotides per dimer.…”

Section: Discussionsupporting

confidence: 88%

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

et al. 2006

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ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.

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Section: Discussionsupporting

confidence: 88%

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

et al. 2006

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“…The observations that amino acids from both NBDs coordinate with each ATP and that around half the area buried at the dimer interface is contributed by the two ATP molecules allow few alternatives to the hypothesis that ATP binding drives 'closed dimer' formation and contributes substantially to its stability [9,10,42]. The precise conformational changes which trigger 'closed dimer' formation remain unclear because structures with and without bound nucleotide have only been obtained for NBDs in the absence of TMDs.…”

Section: Mechanisms Of Transportmentioning

confidence: 99%

“…Biochemical studies of intact transport complexes suggest that the conformational changes at the NBDs are relatively small [43,44], implying a transition between an 'open NBD dimer' and 'closed NBD dimer' configuration rather than a major reorientation of the NBDs with respect to other domains. Free NBDs in solution appear relatively flexible in the absence of ATP [39,42] and cannot form a stable dimer as the buried interface would be relatively small. By comparing the structures of nucleotide-free monomers and ATP-bound dimers (only possible with mutant NBDs with key changes to prevent hydrolysis), it has become clear that high-affinity ATP binding involves 'rigid body' rotation of the α-helical subdomain with respect to the core subdomain (9, 10, 42; Fig.…”

Section: Mechanisms Of Transportmentioning

confidence: 99%

“…Two ATP molecules are normally bound at the dimer interface in the 'closed dimer'-two bound nucleotides have been observed in crystallographic dimers of mutant LolD [10] and HlyB-NBD [51], MalK [42] and Rad50 [9]; have been detected biochemically in the Mdl1p dimer [55]; and have been shown, biochemically, to be bound by the intact MRP1 and P-gp transporters [34,52]. For CFTR, there is indirect evidence that ATP must bind to both sites to open the channel [59,60].…”

Section: Stoichiometry Of Atp Binding and Hydrolysismentioning

confidence: 99%

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Structure and function of ABC transporters: the ATP switch provides flexible control

Linton

Higgins

2006

Pflugers Arch - Eur J Physiol

190

160

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ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that facilitate the transbilayer movement of ligands. They comprise, minimally, two transmembrane domains, which impart ligand specificity, and two nucleotide-binding domains (NBDs), which power the transport cycle. Almost 25 years of biochemistry is reviewed in light of the recent structure analyses resulting in the ATP-switch model for function in which the NBDs switch between a dimeric conformation, closed around two molecules of ATP, and a nucleotide-free, dimeric 'open' conformation. The flexibility of this switching mechanism has evolved to provide different kinetic control for different transporters and has also been co-opted to diverse functions other than transmembrane transport.

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“…Crystallographic structures suggest that the active conformation of the NBDs consists of the Walker A motif from one NBD and the LSGGQ from the second NBD, forming a "nucleotidesandwich" (20). Each NBD is able to bind one ATP, and it is proposed that two ATPs are bound to the homodimer to form the catalytically active closed dimer state as demonstrated by the structures of MJ0796 (18), E. coli MalK (21), and Rad50 (22).…”

mentioning

confidence: 99%

Characterization of the LSGGQ and H Motifs from the Escherichia coli Lipid A Transporter MsbA

Buchaklian

Klug

2006

Biochemistry

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ATP binding cassette (ABC) transporters make up one of the largest superfamilies of proteins known and have been shown to transport substrates ranging from lipids and antibiotics to sugars and amino acids. The dysfunction of ABC transporters has been linked to human pathologies such as cystic fibrosis, hyperinsulinemia, and macular dystrophy. Several bacterial ABC transporters are also necessary for bacterial survival and transport of virulence factors in an infected host. MsbA is a 65kDa protein that forms a functional homodimer consisting of two six-helix transmembrane domains and two approximately 250 amino acid nucleotide binding domains (NBD). The NBDs contain several conserved regions such as the Walker A, LSGGQ and H-motif that bind directly to ATP and align it for hydrolysis. MsbA transports lipid A, its native substrate, across the inner membrane of Gram-negative bacteria. Loss or dysfunction of MsbA results in a toxic accumulation of lipid A inside the cell, leading to cell membrane instability and cell death. Using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, conserved motifs within the MsbA NBD have been evaluated for structure and dynamics upon substrate binding. It has been determined that the LSGGQ NBD consensus sequence is consistent with an α-helical conformation and that these residues maintain extensive tertiary contacts throughout hydrolysis. The dynamics of the LSGGQ and the H-motif region have been studied in the presence of ATP, ADP, and ATP plus vanadate to identify the residues that are directly affected by interactions with the substrate before, after, and during hydrolysis, respectively. ATP binding cassette (ABC) transporters are one of the largest superfamilies of proteins known (1;2). They are classified by their ability to couple the hydrolysis of ATP to the transport of a variety of substrates either into or out of cells (1;3;4). Defects in endogenous human ABC transporters have been implicated in several pathologies including cystic fibrosis, cholestasis, artherosclerosis, hypoglycemia, hyperbiliruginemia, and macular dystrophy and degeneration diseases (3). Also in humans, ABC transporters such as P-glycoprotein are able to transport chemotherapeutic drugs and lipids resulting in the reduced effectiveness of cancer treatments (5;6). ABC transporters in bacteria are not only essential for survival but are also able to secrete toxins and antimicrobial agents. This ability to expel antimicrobial agents may be involved in the development of multidrug resistance (MDR) (7-13). MDR has become a serious problem facing the medical community today, and its impact can be seen in the treatment of diseases ranging from cancer to human immunodeficiency virus (HIV), to numerous bacterial infections. These facts make ABC transporters an important target in the fight against MDR. By studying these transporters in detail it may be possible to identify new and novel agents to combat infections by pathogens. Bacterial efflux ABC transporters transpo...

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A Tweezers-like Motion of the ATP-Binding Cassette Dimer in an ABC Transport Cycle

Cited by 471 publications

References 47 publications

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

Structure and function of ABC transporters: the ATP switch provides flexible control

Characterization of the LSGGQ and H Motifs from the Escherichia coli Lipid A Transporter MsbA

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