Interdependence of calcium and cobalamin binding by wild-type and mutant BtuB protein in the outer membrane of Escherichia coli.
The binding of calcium and cobalamin to outer membranes from cells of Escherichia coli that contained amplified levels of wild-type or mutant btuB was studied. The mutant (BBam50) had an aspartyl-prolyl dipeptide inserted after the original 50th amino acid residue of the mature BtuB protein, which is within a region that shows extensive homology with the ferric siderophore receptors. This insertion resulted in cleavage of the BtuB in two places. The larger form retained the insertion but had lost 11 amino acid residues from the amino terminus. The smaller form was cut at the insertion site. Both the wild-type protein and the larger form of mutant BtuB showed calcium-dependent cobalamin binding with the same affinity for cobalamin, although the mutant had a much lower affinity for calcium. The smaller form of the mutant BtuB protein had a greatly reduced affinity for cobalamin, which was probably the result of inactivation of the cobalamin-dependent calcium-binding site. Cobalamin-dependent calcium binding was measured in wild-type BtuB preparations and was found to have the same corrinoid specificity and response to various corrinoid concentrations as shown previously for cobalamin binding. The results are consistent with a role for calcium in the cobalamin pump of the outer membrane of E. coli and show that a conserved part of the BtuB protein is required for the cobalamin-dependent binding of calcium.
Bradbeer
C
C
Department of Biochemistry, University of Virginia School of Medicine, Charlottesville 22908.
Gudmundsdottir
A
A
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide; D014805:Vitamin B 12 / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D014805:Vitamin B 12; D002118:Calcium