title: Probing the conformation of the resting state of a bacterial multidrug ABC transporter, BmrA, by a site-directed spin labeling approach.
abstract: Previously published 3-D structures of a prototypic ATP-binding cassette (ABC) transporter, MsbA, have been recently corrected revealing large rigid-body motions possibly linked to its catalytic cycle. Here, a closely related multidrug bacterial ABC transporter, BmrA, was studied using site-directed spin labeling by focusing on a region connecting the transmembrane domain and the nucleotide-binding domain (NBD). Electron paramagnetic resonance (EPR) spectra of single spin-labeled cysteine mutants suggests that, in the resting state, this sub-domain essentially adopts a partially extended conformation, which is consistent with the crystal structures of MsbA and Sav1866. Interestingly, one of the single point mutants (Q333C) yielded an immobilized EPR spectrum that could arise from a direct interaction with a vicinal tyrosine residue. Inspection of different BmrA models pointed to Y408, within the NBD, as the putative interacting partner, and its mutation to a Phe residue indeed dramatically modified the EPR spectra of the spin labeled Q333C. Moreover, unlike the Y408F mutation, the Y408A mutation abolished both ATPase activity and drug transport of BmrA, suggesting that a nonpolar bulky residue is required at this position. The spatial proximity of Q333 and Y408 was also confirmed by formation of a disulfide bond when both Q333 and T407 (or S409) were replaced jointly by a cysteine residue. Overall, these results indicate that the two regions surrounding Q333 and Y408 are close together in the 3-D structure of BmrA and that residues within these two sub-domains are essential for proper functioning of this transporter.
authors.affiliation: Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-Université Lyon I and IFR 128, 7 passage du Vercors, 69367 Lyon Cedex 07, France.
authors.forename: Marie-Ange
authors.initials: MA
authors.lastname: Do Cao
authors.forename: Serge
authors.initials: S
authors.lastname: Crouzy
authors.forename: Miyeon
authors.initials: M
authors.lastname: Kim
authors.forename: Michel
authors.initials: M
authors.lastname: Becchi
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
authors.forename: Attilio
authors.initials: A
authors.lastname: Di Pietro
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Jault
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D011487:Protein Conformation; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013113:Spin Labels
chemical_list: D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; D013113:Spin Labels; D003545:Cysteine