title: EPR spectroscopy of MolB2C2-a reveals mechanism of transport for a bacterial type II molybdate importer.
abstract: In bacteria, ATP-binding cassette (ABC) transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know has been observed in studies of the vitamin B12 importer BtuC2D2. MolB2C2 (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is ∼10-fold smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC, for which alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and EPR spectroscopy. From these studies, we found that nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformational changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as "gate I," remains open throughout the MolBC-A mechanism, and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.
authors.affiliation: From the Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208.
authors.forename: Austin J
authors.initials: AJ
authors.lastname: Rice
authors.affiliation: the Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, and.
authors.forename: Frances J D
authors.initials: FJD
authors.lastname: Alvarez
authors.affiliation: the Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-0509.
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Schultz
authors.affiliation: the Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-0509.
authors.forename: Candice S
authors.initials: CS
authors.lastname: Klug
authors.affiliation: the Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, and.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Davidson
authors.affiliation: From the Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208,. Electronic address: h-pinkett@northwestern.edu.
authors.forename: Heather W
authors.initials: HW
authors.lastname: Pinkett
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006193:Haemophilus influenzae / Q000378:metabolism*; D008024:Ligands; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008954:Models, Biological; D008982:Molybdenum / Q000378:metabolism*; D019897:Periplasm / Q000187:drug effects / Q000378:metabolism; D017433:Protein Structure, Secondary
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D008024:Ligands; D026901:Membrane Transport Proteins; C044659:molybdate; D008982:Molybdenum; D000255:Adenosine Triphosphate
keywords: ABC Transporter; Electron Paramagnetic Resonance (EPR); Importer; Membrane Proteins; Molybdate; Protein Conformation; Protein Dynamics