The Q-loop Disengages from the First Intracellular Loop during the Catalytic Cycle of the Multidrug ABC Transporter BmrA - Groupe Entrée et bourgeonnement des virus à enveloppe / Entry and Budding of Enveloped Viruses Group (EBEV) Access content directly
Journal Articles Journal of Biological Chemistry Year : 2005

The Q-loop Disengages from the First Intracellular Loop during the Catalytic Cycle of the Multidrug ABC Transporter BmrA

Abstract

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, conforma-tional changes of BmrA, a multidrug 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 vana-date 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-link-ers separated by more than 13 Å were added. Altogether, these results give support to the existence, in the resting state, of a mono-meric 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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Dates and versions

hal-01988190 , version 1 (28-01-2019)

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Olivier Dalmas, Cedric Orelle, Anne-Emmanuelle Foucher, Christophe Geourjon, Serge Crouzy, et al.. The Q-loop Disengages from the First Intracellular Loop during the Catalytic Cycle of the Multidrug ABC Transporter BmrA. Journal of Biological Chemistry, 2005, 280 (44), pp.36857-36864. ⟨10.1074/jbc.m503266200⟩. ⟨hal-01988190⟩
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