Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter

Orelle, Cédric; Ayvaz, Tulin; Everly, R. Michael; Klug, Candice S.; Davidson, Amy L.

doi:10.1073/pnas.0803799105

Cited by 95 publications

(196 citation statements)

References 36 publications

(41 reference statements)

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“…In the absence of maltose, the transporter rests in a catalytically incompetent conformation due to the separation of the LSGGQ motif from the active site (5). Binding of maltose-loaded MBP and ATP are both required to bring about the conformational changes that result in the opening of MBP for delivery of maltose to the translocation cavity and the closure of the MalK dimer for the hydrolysis of ATP (34,35).…”

Section: Discussionmentioning

confidence: 99%

Snapshots of the maltose transporter during ATP hydrolysis

Oldham

Chen

2011

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

183

222

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ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5′-(β,γ-imido)triphosphate or ADP in conjunction with phosphate analogs BeF 3 − , VO 4 3− , or AlF 4 − , were determined to 2.2-to 2.4-Å resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.membrane protein | transition state | ground state A TP-binding cassette (ABC) transporters are large membrane protein complexes powered by ATP (1). In prokaryotes, ABC transporters are survival factors mediating the uptake of nutrients and the efflux of antimicrobial agents and virulence factors. In humans, 48 ABC transporters are identified that transport a variety of compounds and are responsible for diseases including cystic fibrosis, cholestasis, and multidrug resistance in cancer (2). ABC transporters, both importers and exporters, contain two transmembrane domains (TMDs) that form a substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Structures of intact transporters show that an inward-facing state, where the substrate translocation pathway is accessible from the cytoplasm, coincides with an open NBD dimer in which the ATPase active sites are separated (3-8). Closure of the NBD dimer in the presence of ATP is concomitant with reorientation of the substrate translocation pathway from an inward-facing to an outward-facing configuration (7, 9, 10). Several sequences are highly conserved among NBDs for ATP hydrolysis, including: (I) the Walker A motif with a consensus sequence of GxxGxGKST, where x represents any amino acid; (ii) the Walker B motif, which has four hydrophobic residues followed by a negatively charged residue; (iii) the D loop, containing a conserved aspartic acid; (iv) the LSGGQ loop, or the signature motif, diagnostic of ABC ATPases; (v) the Q loop, named after its invariable glutamine; and (vi) the switch region that contains a highly conserved histidine residue. However, uncertainty exists regarding the functional role of these motifs and the chemistry of ATP hydrolysis. For example, based on studies of isolated NBDs, the acidic residue immediately following the Walker B motif, a glutamate in most ABC transporters, was suggested by several groups to act as a general base to polarize the hydrolytic water molecule (11,12). But in a different model, this glutamate residue functions, instead, to orient the switch histidine while the ATP is hydrolyzed via substrate-assisted catalysis rather than by a general base mechanism (13). High-resolution structures of catalytic intermediates of a complete ABC transporter would be valuable to establish the chemical mechanism of ATP hydr...

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Section: Discussionmentioning

confidence: 99%

Snapshots of the maltose transporter during ATP hydrolysis

Oldham

Chen

2011

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

183

222

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“…Plasmids carrying mutation(s) encoding the cysteine substitutions in the malK gene were used to transform an E. coli strain containing a chromosomal deletion of malK, and were tested for function by complementation, as judged by the red color of the colonies on maltose MacConkey agar plates (22). By this criterion, all of the substitutions in the helical and RecA-like subdomains were functional, but only one of the Q-loop substitutions (S83C) was functional.…”

Section: Positioning Spin Labels To Detect α-Helical Subdomain Rotatimentioning

confidence: 99%

“…The maltose transporter has been crystallized in an inwardfacing conformation in the absence of nucleotide (23), and an outward-facing conformational intermediate, stabilized by binding of both ATP and MBP, that may resemble the transition state (22,24,25) (Fig. 1A).…”

Section: Positioning Spin Labels To Detect α-Helical Subdomain Rotatimentioning

confidence: 99%

“…1B) were also mutated to cysteines, individually and in pairs, to measure changes in the distance between these two subdomains upon rotation. Residues were selected for mutation based on distance constraints (28, 29) and side-chain solvent-accessibility [http://mobyle.rpbs.univparis-diderot.fr/cgi-bin/portal.py?form=ASA (30)], to maximize spin-labeling efficiency.Plasmids carrying mutation(s) encoding the cysteine substitutions in the malK gene were used to transform an E. coli strain containing a chromosomal deletion of malK, and were tested for function by complementation, as judged by the red color of the colonies on maltose MacConkey agar plates (22). By this criterion, all of the substitutions in the helical and RecA-like subdomains were functional, but only one of the Q-loop substitutions (S83C) was functional.…”

mentioning

confidence: 99%

“…A periplasmic maltose-binding protein (MBP) delivers the substrate to the transporter and stimulates its ATPase activity (21). In the maltose transporter, closure of the NBD dimer requires both ATP and MBP (22) and involves two separate rotational events, a ∼10°rotation of the α-helical subdomains relative to the RecA-like subdomains and a ∼14°rotation of the RecA-like subdomains relative to the C-terminal regulatory domains (23). Analysis of the helical subdomain rotation is crucial for a detailed understanding of how ATP hydrolysis is stimulated by MBP and coupled to transport.…”

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confidence: 99%

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Dynamics of α-helical subdomain rotation in the intact maltose ATP-binding cassette transporter

Orelle

Alvarez²,

Oldham³

et al. 2010

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

Self Cite

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ATP-binding cassette (ABC) transporters are powered by a nucleotide-binding domain dimer that opens and closes during cycles of ATP hydrolysis. These domains consist of a RecA-like subdomain and an α-helical subdomain that is specific to the family. Many studies on isolated domains suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP binding, moving the family signature motif into a favorable position to interact with the nucleotide across the dimer interface. Moreover, the transmembrane domains are docked into a cleft at the interface between these subdomains, suggesting a putative role of the rotation in interdomain communication. Electron paramagnetic resonance spectroscopy was used to study the dynamics of this rotation in the intact Escherichia coli maltose transporter MalFGK 2 . This importer requires a periplasmic maltose-binding protein (MBP) that activates ATP hydrolysis by promoting the closure of the cassette dimer (MalK 2 ). Whereas this rotation occurred during the transport cycle, it required not only trinucleotide, but also MBP, suggesting it is part of a global conformational change in the transporter. Interaction of AMP-PNP-Mg 2þ and a MBP that is locked in a closed conformation induced a transition from open MalK 2 to semiopen MalK 2 without significant subdomain rotation. Inward rotation of the helical subdomain and complete closure of MalK 2 therefore appear to be coupled to the reorientation of transmembrane helices and the opening of MBP, events that promote transfer of maltose into the transporter. After ATP hydrolysis, the helical subdomain rotates out as MalK 2 opens, resetting the transporter in an inward-facing conformation.EPR spectroscopy | transport mechanism | membrane protein A TP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies in organisms, and mediate the translocation of a wide range of substrates across the membrane (1). These transporters typically contain two transmembrane domains (TMDs) and are energized by a nucleotide-binding domain (NBD) dimer that closes and opens during cycles of ATP binding and hydrolysis (2, 3). Each NBD consists of a RecA-like subdomain, found in numerous ATPases (4), and an α-helical subdomain that is specific to the ABC family (5). Crystallographic studies (5-8) and molecular dynamic simulations (9, 10), performed on isolated NBDs, suggest that the helical subdomain rotates toward the RecA-like subdomain in response to ATP binding. This rotation positions the ABC family signature motif to interact with nucleotide across the dimer interface so that the NBDs can close to hydrolyze ATP. After ATP hydrolysis, it is suggested that the helical subdomain rotates away (5, 11). In addition, the TMDs contact the NBDs at the interface between these subdomains (12-15), suggesting a putative role of the rotation in interdomain communication (16). Here, we used sitedirected spin labeling electron paramagnetic resonance (EPR) spectroscopy (17,18) to study the dynamics of this rotation...

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Structures of ABC transporters: handle with care

2020

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In the past two decades, the ATP-binding cassette (ABC) transporters' field has undergone a structural revolution. The importance of structural biology to the development of the field of ABC transporters cannot be overstated, as the ensemble of structures not only revealed the architecture of ABC transporters but also shaped our mechanistic view of these remarkable molecular machines. Nevertheless, we advocate that the mechanistic interpretation of the structures is not trivial and should be carried out with prudence. Herein, we bring several examples of structures of ABC transporters that merit re-interpretation via careful comparison to experimental data. We propose that it is of the upmost importance to place new structures within the context of the available experimental data.

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Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter

Abstract: catalytic cycle ͉ conformational change ͉ EPR ͉ membrane protein ͉ P-glycoprotein

Cited by 95 publications

References 36 publications

Snapshots of the maltose transporter during ATP hydrolysis

Snapshots of the maltose transporter during ATP hydrolysis

Dynamics of α-helical subdomain rotation in the intact maltose ATP-binding cassette transporter

Structures of ABC transporters: handle with care

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