scholarly journals Crosslinking of nucleotide binding domains improves the coupling efficiency of an ABC transporter

2019 ◽  
Author(s):  
Chengcheng Fan ◽  
Jens T. Kaiser ◽  
Douglas C. Rees
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Atp Hydrolysis ◽  
Conformational Dynamics ◽  
Coupling Efficiency ◽  
Nucleotide Binding ◽  
Disulfide Crosslinking ◽  
Binding Domains ◽  
Efficient Coupling ◽  
The Impact

AbstractATP Binding Cassette (ABC) transporters often exhibit significant basal ATPase activity in the absence of transported substrates. To investigate the factors that contribute to this inefficient coupling of ATP hydrolysis to transport, we characterized the structures and functions of variants of the bacterial Atm1 homolog from Novosphingobium aromaticivorans (NaAtm1), including forms with disulfide crosslinks between the nucleotide binding domains. Unexpectedly, disulfide crosslinked variants of NaAtm1 reconstituted into proteoliposomes not only transported oxidized glutathione, but also exhibited more efficient coupling of ATP hydrolysis to GSSG transport than the native transporter. These observations suggest that enhanced conformational dynamics of reconstituted NaAtm1 may contribute to the inefficient use of ATP. Understanding the origins of this uncoupled ATPase activity, and reducing the impact through disulfide crosslinking or other protocols, will be critical for the detailed dissection of ABC transporter mechanism to assure that the ATP dependent steps are indeed relevant to substrate translocation.

scholarly journals The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer

2006 ◽  
Vol 401 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Fiona L. L. Stratford ◽  
Mohabir Ramjeesingh ◽  
Joanne C. Cheung ◽  
Ling-JUN Huan ◽  
Christine E. Bear
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Vital Role ◽  
Atp Binding ◽  
Primary Role ◽  
Binding Domains ◽  
Membrane Spanning ◽  
Atp Binding And Hydrolysis

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

scholarly journals Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

2001 ◽  
Vol 183 (16) ◽  
pp. 4761-4770 ◽  
Author(s):  
Juan M. Falcón-Pérez ◽  
Mónica Martı́nez-Burgos ◽  
Jesús Molano ◽  
Marı́a J. Mazón ◽  
Pilar Eraso
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Transmembrane Domain ◽  
Substrate Binding ◽  
Nucleotide Binding ◽  
Intramolecular Interactions ◽  
Phenotypic Characterization ◽  
Atp Binding ◽  
Binding Domains ◽  
Atp Binding Cassette

ABSTRACT The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V max of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.

scholarly journals Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Smriti Mishra ◽  
Brandy Verhalen ◽  
Richard A Stein ◽  
Po-Chao Wen ◽  
Emad Tajkhorshid ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Conformational Dynamics ◽  
Nucleotide Binding ◽  
Power Stroke ◽  
Spin Labels ◽  
Nucleotide Binding Sites ◽  
Binding Domains ◽  
Cytotoxic Molecules ◽  
Asymmetric Configuration

Multidrug ATP binding cassette (ABC) exporters are ubiquitous ABC transporters that extrude cytotoxic molecules across cell membranes. Despite recent progress in structure determination of these transporters, the conformational motion that transduces the energy of ATP hydrolysis to the work of substrate translocation remains undefined. Here, we have investigated the conformational cycle of BmrCD, a representative of the heterodimer family of ABC exporters that have an intrinsically impaired nucleotide binding site. We measured distances between pairs of spin labels monitoring the movement of the nucleotide binding (NBD) and transmembrane domains (TMD). The results expose previously unobserved structural intermediates of the NBDs arising from asymmetric configuration of catalytically inequivalent nucleotide binding sites. The two-state transition of the TMD, from an inward- to an outward-facing conformation, is driven exclusively by ATP hydrolysis. These findings provide direct evidence of divergence in the mechanism of ABC exporters.

scholarly journals Vanadate-Induced Trapping of Nucleotides by Purified Maltose Transport Complex Requires ATP Hydrolysis

2000 ◽  
Vol 182 (23) ◽  
pp. 6570-6576 ◽  
Author(s):  
Susan Sharma ◽  
Amy L. Davidson
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Maltose Binding Protein ◽  
High Energy ◽  
Maltose Transport ◽  
Binding Domains ◽  
Mechanism Of Inhibition ◽  
Transport Complex

ABSTRACT The maltose transport system in Escherichia coli is a member of the ATP-binding cassette superfamily of transporters that is defined by the presence of two nucleotide-binding domains or subunits and two transmembrane regions. The bacterial import systems are unique in that they require a periplasmic substrate-binding protein to stimulate the ATPase activity of the transport complex and initiate the transport process. Upon stimulation by maltose-binding protein, the intact MalFGK2 transport complex hydrolyzes ATP with positive cooperativity, suggesting that the two nucleotide-binding MalK subunits interact to couple ATP hydrolysis to transport. The ATPase activity of the intact transport complex is inhibited by vanadate. In this study, we investigated the mechanism of inhibition by vanadate and found that incubation of the transport complex with MgATP and vanadate results in the formation of a stably inhibited species containing tightly bound ADP that persists after free vanadate and nucleotide are removed from the solution. The inhibited species does not form in the absence of MgCl2 or of maltose-binding protein, and ADP or another nonhydrolyzable analogue does not substitute for ATP. Taken together, these data conclusively show that ATP hydrolysis must precede the formation of the vanadate-inhibited species in this system and implicate a role for a high-energy, ADP-bound intermediate in the transport cycle. Transport complexes containing a mutation in a single MalK subunit are still inhibited by vanadate during steady-state hydrolysis; however, a stably inhibited species does not form. ATP hydrolysis is therefore necessary, but not sufficient, for vanadate-induced nucleotide trapping.

Mg2+-dependent ATP occlusion at the first nucleotide-binding domain (NBD1) of CFTR does not require the second (NBD2)

2008 ◽  
Vol 416 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Luba Aleksandrov ◽  
Andrei Aleksandrov ◽  
John R. Riordan
Keyword(s):  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Atp Binding ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Bivalent Cation ◽  
Binding Domains ◽  
Rapid Hydrolysis ◽  
Cystic Fibrosis Transmembrane

ATP binding to the first and second NBDs (nucleotide-binding domains) of CFTR (cystic fibrosis transmembrane conductance regulator) are bivalent-cation-independent and -dependent steps respectively [Aleksandrov, Aleksandrov, Chang and Riordan (2002) J. Biol. Chem. 277, 15419–15425]. Subsequent to the initial binding, Mg2+ drives rapid hydrolysis at the second site, while promoting non-exchangeable trapping of the nucleotide at the first site. This occlusion at the first site of functional wild-type CFTR is somewhat similar to that which occurs when the catalytic glutamate residues in both of the hydrolytic sites of P-glycoprotein are mutated, which has been proposed to be the result of dimerization of the two NBDs and represents a transient intermediate formed during ATP hydrolysis [Tombline and Senior (2005) J. Bioenerg. Biomembr. 37, 497–500]. To test the possible relevance of this interpretation to CFTR, we have now characterized the process by which NBD1 occludes [32P]N3ATP (8-azido-ATP) and [32P]N3ADP (8-azido-ADP). Only N3ATP, but not N3ADP, can be bound initially at NBD1 in the absence of Mg2+. Despite the lack of a requirement for Mg2+ for ATP binding, retention of the NTP at 37 °C was dependent on the cation. However, at reduced temperature (4 °C), N3ATP remains locked in the binding pocket with virtually no reduction over a 1 h period, even in the absence of Mg2+. Occlusion occurred identically in a ΔNBD2 construct, but not in purified recombinant NBD1, indicating that the process is dependent on the influence of regions of CFTR in addition to NBD1, but not NBD2.

scholarly journals Association/Dissociation of the Nucleotide-binding Domains of the ATP-binding Cassette Protein MsbA Measured during Continuous Hydrolysis

2013 ◽  
Vol 288 (29) ◽  
pp. 20785-20796 ◽  
Author(s):  
Rebecca S. Cooper ◽  
Guillermo A. Altenberg
Keyword(s):  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Binding Domains ◽  
Partial Opening ◽  
Contact Models ◽  
Bacterial Lipid ◽  
Atp Binding Cassette

In ATP-binding cassette proteins, the two nucleotide-binding domains (NBDs) work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. It is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or partial opening of the dimers such that the NBDs remain in contact during the hydrolysis cycle. We studied the bacterial lipid flippase MsbA by luminescence resonance energy transfer (LRET). The LRET signal between optical probes reacted with single-cysteine mutants was employed to follow NBD association/dissociation in real time. The intermonomer distances calculated from LRET data indicate that the NBDs separate completely following ATP hydrolysis, even in the presence of mm MgATP, and that the dissociation occurs following each hydrolysis cycle. The results support association/dissociation, as opposed to constant contact models, for the mode of operation of ATP-binding cassette proteins.

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