title: Conformational cycle of the vitamin B12 ABC importer in liposomes detected by double electron-electron resonance (DEER).
abstract: Double electron-electron resonance is used here to investigate intermediates of the transport cycle of the Escherichia coli vitamin B12 ATP-binding cassette importer BtuCD-F. Previously, we showed the ATP-induced opening of the cytoplasmic gate I in TM5 helices, later confirmed by the AMP-PNP-bound BtuCD-F crystal structure. Here, other key residues are analyzed in TM10 helices (positions 307 and 322) and in the cytoplasmic gate II, i.e. the loop between TM2 and TM3 (positions 82 and 85). Without BtuF, binding of ATP induces detectable changes at positions 307 and 85 in BtuCD in liposomes. Together with BtuF, ATP triggers the closure of the cytoplasmic gate II in liposomes (reported by both positions 82 and 85). This forms a sealed cavity in the translocation channel in agreement with the AMP-PNP·BtuCD-F x-ray structure. When vitamin B12 and AMP-PNP are simultaneously present, the extent of complex formation is reduced, but the short 82-82 interspin distance detected indicates that the substrate does not affect the closed conformation of this gate. The existence of the BtuCD-F complex under these conditions is verified with spectroscopically orthogonal nitroxide and Gd(III)-based labels. The cytoplasmic gate II remains closed also in the vanadate-trapped state, but it reopens in the ADP-bound state of the complex. Therefore, we suggest that the substrate likely trapped in ATP·BtuCD-F can be released after ATP hydrolysis but before the occluded ADP-bound conformation is reached.
authors.affiliation: From the Laboratory of Physical Chemistry, ETH Zurich, Wolfgang-Pauli-Strasse 10 and.
authors.forename: Benesh
authors.initials: B
authors.lastname: Joseph
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Korkhov
authors.forename: Maxim
authors.initials: M
authors.lastname: Yulikov
authors.forename: Gunnar
authors.initials: G
authors.lastname: Jeschke
authors.forename: Enrica
authors.initials: E
authors.lastname: Bordignon
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D014805:Vitamin B 12 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; C050021:BtuC peptide, E coli; C050022:BtuD peptide, E coli; D029968:Escherichia coli Proteins; D033902:Periplasmic Binding Proteins; C446048:btuF protein, E coli; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014805:Vitamin B 12
keywords: ABC Transporter; DEER; Detergent; Electron Paramagnetic Resonance (EPR); Liposomes; Membrane Bilayer; Membrane Proteins; Peldor