Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis

The sequence of the gyrase B subunit gene from Staphylococcus aureus strains resistant to the gyrase B subunit inhibitors cyclothialidine, coumermycin, and novobiocin has been determined. The residues altered in the resistant gyrase B subunits map to the ATP-binding region, suggesting that the drugs inhibit ATP binding and hydrolysis. The pattern of cross-resistances indicates that the detailed binding mode of the compounds differs.

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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

Biochemical Journal ◽

10.1042/bj20060968 ◽

2006 ◽

Vol 401 (2) ◽

pp. 581-586 ◽

Cited By ~ 29

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.

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In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012042 ◽

2020 ◽

Vol 295 (15) ◽

pp. 5002-5011 ◽

Cited By ~ 4

Author(s):

Ryota Futamata ◽

Fumihiko Ogasawara ◽

Takafumi Ichikawa ◽

Atsushi Kodan ◽

Yasuhisa Kimura ◽

...

Keyword(s):

Conformational Changes ◽

Drug Transport ◽

Atp Hydrolysis ◽

Hek293 Cells ◽

Atp Binding ◽

Binding Domains ◽

P Glycoprotein ◽

Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

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Learning the ABCs one at a time: structure and mechanism of ABC transporters

Biochemical Society Transactions ◽

10.1042/bst20180147 ◽

2019 ◽

Vol 47 (1) ◽

pp. 23-36 ◽

Cited By ~ 36

Author(s):

Robert C. Ford ◽

Konstantinos Beis

Keyword(s):

Abc Transporters ◽

Atp Hydrolysis ◽

Atp Binding ◽

Bacterial Cells ◽

The Novel ◽

Mechanism Model ◽

Binding Domains ◽

Atp Binding And Hydrolysis ◽

Alternating Access

Abstract ATP-binding cassette (ABC) transporters are essential proteins that are found across all kingdoms of life. ABC transporters harness the energy of ATP hydrolysis to drive the import of nutrients inside bacterial cells or the export of toxic compounds or essential lipids across bacteria and eukaryotic membranes. Typically, ABC transporters consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) to bind their substrate and ATP, respectively. The TMDs dictate what ligands can be recognised, whereas the NBDs are the power engine of the ABC transporter, carrying out ATP binding and hydrolysis. It has been proposed that they utilise the alternating access mechanism, inward- to outward-facing conformation, to transport their substrates. Here, we will review the recent progress on the structure determination of eukaryotic and bacterial ABC transporters as well as the novel mechanisms that have also been proposed, that fall out of the alternating access mechanism model.

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