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

Pretz, Monika G.; Albers, Sonja‐Verena; Schuurman-Wolters, Gea K.; Tampé, Robert; Driessen, Arnold J. M.; Does, Chris van der

doi:10.1021/bi061230e

Cited by 14 publications

(26 citation statements)

References 44 publications

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“…The two Walker A BmrA mutants described here are still able to bind both ATP-Mg and the 8-N 3 -ATP nucleotide analogue, albeit with a seemingly reduced efficiency, and this is in agreement with the results found after mutation of the equivalent Lys residue in other ABC transporters (30,38,(51)(52)(53). Mounting evidence supports the view that, in all native ABC transporters, the ATP-Mg binding step triggers the dimerization of the two NBDs in a transient conformation where two ATP molecules are tightly sandwiched at the NBDs interface (9,12,45,47,(54)(55)(56). While this certainly holds true for wild-type BmrA, our data suggest that this ATP-Mg dependent dimerization step is impaired in the Walker A mutants (see Figure 6), for the following reasons: (i) In contrast to wild-type BmrA, the K380R mutant hydrolyzes ATP according to Michaelis-Menten kinetics.…”

Section: Discussionsupporting

confidence: 87%

Conformational Change Induced by ATP Binding in the Multidrug ATP-Binding Cassette Transporter BmrA

et al. 2008

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ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.

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Section: Discussionsupporting

confidence: 87%

Conformational Change Induced by ATP Binding in the Multidrug ATP-Binding Cassette Transporter BmrA

et al. 2008

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“…Indeed, this is expected, because Pgp has an uncoupled cycle and can hydrolyze ATP efficiently in the absence of drugs. In addition, a thermodynamic study of isolated NBD subunits of a bacterial ABC protein showed that NBD dimer formation is itself a facile and energetically favorable reaction in the absence of the transmembrane domains and transport substrate (62). In Fig.…”

Section: Discussionmentioning

confidence: 99%

Characterization of an Asymmetric Occluded State of P-glycoprotein with Two Bound Nucleotides

Siarheyeva

Liu

Sharom

2010

Journal of Biological Chemistry

103

110

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P-glycoprotein (ABCB1), a member of the ABC superfamily, functions as an ATP-driven multidrug efflux pump. The catalytic cycle of ABC proteins is believed to involve formation of a sandwich dimer in which two ATP molecules are bound at the interface of the nucleotide binding domains (NBDs). However, such dimers have only been observed in isolated NBD subunits and catalytically arrested mutants, and it is still not understood how ATP hydrolysis is coordinated between the two NBDs. We report for the first time the characterization of an asymmetric state of catalytically active native P-glycoprotein with two bound molecules of adenosine 5-(␥-thio)triphosphate (ATP␥S), one of low affinity (K d 0.74 mM), and one "occluded" nucleotide of 120-fold higher affinity (K d 6 M). ATP␥S also interacts with P-glycoprotein with high affinity as assessed by inhibition of ATP hydrolysis and protection from covalent labeling of a Walker A Cys residue, whereas other non-hydrolyzable ATP analogues do not. Binding of ATP␥S (but not ATP) causes Trp residue heterogeneity, as indicated by collisional quenching, suggesting that it may induce conformational asymmetry. Asymmetric ATP␥S-bound P-glycoprotein does not display reduced binding affinity for drugs, implying that transport is not driven by ATP binding and likely takes place at a later stage of the catalytic cycle. We propose that this asymmetric state with two bound nucleotides represents the next intermediate on the path toward ATP hydrolysis after nucleotide binding, and an alternating sites mode of action is achieved by simultaneous switching of the two active sites between high and low affinity states.

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“…3 C, black). In contrast, we assumed a right-shifted equilibrium for step C 3 –C 4 (ΔG C4–C3 = −4.0 k T), consistent with the exergonic nature of ATP binding (Pretz et al, 2006). Finally, using standard Q-matrix techniques (Colquhoun and Sigworth, 1995), the rates for the C 1 –C 3 equilibrium were chosen to yield a K Po of 59 µM (compare Fig.…”

Section: Discussionmentioning

confidence: 99%

Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2

Szöllősi

Vergani

Csanády

2010

Journal of General Physiology

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The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dimer drives the opening of the chloride ion pore, little is known about how ATP binding to individual NBDs promotes subsequent formation of this stable dimer. Structural studies on isolated NBDs suggest that ATP binding induces an intra-domain conformational change termed “induced fit,” which is required for subsequent dimerization. We investigated the allosteric interaction between three residues within NBD2 of CFTR, F1296, N1303, and R1358, because statistical coupling analysis suggests coevolution of these positions, and because in crystal structures of ABC domains, interactions between these positions appear to be modulated by ATP binding. We expressed wild-type as well as F1296S, N1303Q, and R1358A mutant CFTR in Xenopus oocytes and studied these channels using macroscopic inside-out patch recordings. Thermodynamic mutant cycles were built on several kinetic parameters that characterize individual steps in the gating cycle, such as apparent affinities for ATP, open probabilities in the absence of ATP, open probabilities in saturating ATP in a mutant background (K1250R), which precludes ATP hydrolysis, as well as the rates of nonhydrolytic closure. Our results suggest state-dependent changes in coupling between two of the three positions (1296 and 1303) and are consistent with a model that assumes a toggle switch–like interaction pattern during the intra-NBD2 induced fit in response to ATP binding. Stabilizing interactions of F1296 and N1303 present before ATP binding are replaced by a single F1296-N1303 contact in ATP-bound states, with similar interaction partner toggling occurring during the much rarer ATP-independent spontaneous openings.

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

Cited by 14 publications

References 44 publications

Conformational Change Induced by ATP Binding in the Multidrug ATP-Binding Cassette Transporter BmrA

Conformational Change Induced by ATP Binding in the Multidrug ATP-Binding Cassette Transporter BmrA

Characterization of an Asymmetric Occluded State of P-glycoprotein with Two Bound Nucleotides

Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2

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