The Q-loop disengages from the first intracellular loop during the catalytic cycle of the multidrug ABC transporter BmrA

被引:43
作者
Dalmas, O
Orelle, C
Foucher, AE
Geourjon, C
Crouzy, S
Di Pietro, A
Jault, JM
机构
[1] Commiss Energie Atom, UJF, CNRS,Unite Mixte Rech 5090, DRDC,Lab Biophys Mol & Cellulaire, F-38054 Grenoble, France
[2] Inst Biol & Chim Prot, CNRS, Unite Mixte Rech 5086, UCBL 1, F-69367 Lyon, France
[3] Inst Biol & Chim Prot, CNRS, Unite Mixte Rech 5086, IFR 128, F-69367 Lyon, France
关键词
D O I
10.1074/jbc.M503266200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, amultidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide- binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 angstrom were added. Altogether, these results give support to the existence, in the resting state, of amonomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide- binding domain for the proper functioning of a multidrug ATP-binding cassette transporter.
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页码:36857 / 36864
页数:8
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