title: Observations on the calcium dependence and reversibility of cobalamin transport across the outer membrane of Escherichia coli.
abstract: The calcium dependence of cobalamin (Cbl) binding to the BtuB protein of Escherichia coli and the reversibility of its function in the transport of Cbl across the outer membrane have been examined. The results show that the two calcium-binding sites in BtuB that were identified previously by others are responsible for the calcium dependence of high affinity Cbl binding. The affinity of the pure BtuB protein for Cbl was approximately 1000-fold higher in the presence of saturating levels of calcium than in its absence. The affinities of BtuB for both Cbl and calcium were decreased by insertion of alanine residues at position 51 of the mature protein and were increased by several mutations and deletions in the TonB box. Experiments on the uptake of Cbl into the periplasmic space showed that this process is reversible and that the exit of Cbl back into the medium does not require the protonmotive force. Our interpretation of these results is that the role of the TonB-ExbB-ExbD complex, potentiated by the protonmotive force, is to reduce the affinity of the Cbl-binding site, thus increasing the rate of Cbl release into the periplasmic space. The evidence also indicates that access of the Cbl-binding site of BtuB to the periplasmic space does not require removal of the hatch domain from the barrel.
authors.affiliation: Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908-0733, USA.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Cadieux
authors.forename: Nazir
authors.initials: N
authors.lastname: Barekzi
authors.forename: Clive
authors.initials: C
authors.lastname: Bradbeer
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D002118:Calcium / Q000737:chemistry*; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014805:Vitamin B 12 / Q000737:chemistry*
chemical_list: D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C489181:tonB protein, E coli; D014805:Vitamin B 12; D002118:Calcium