title: Demonstration of the functional role of conserved Glu-Arg residues in the Staphylococcus aureus ferrichrome transporter.
abstract: The features that govern the interaction of ligand binding proteins with membrane permeases of cognate ABC transporters are largely unknown. Using sequence alignments and structural modeling based on the structure of the Escherichia coli BtuCD vitamin B12 transporter, we identified six conserved basic residues in the permease, comprised of FhuB and FhuG proteins, in the ferrichrome transporter of Staphylococcus aureus. Using alanine-scanning mutagenesis we demonstrate that two of these residues, FhuB Arg-71 and FhuG Arg-61, play a more dominant role in transporter function than FhuB Arg-74 and Arg-311, and FhuG Arg-64 and Lys-306. Moreover, we show that at positions 71 and 61 in FhuB and FhuG, respectively, arginine cannot be substituted for lysine without loss of transporter function. Previously, our laboratory demonstrated the importance of conserved acidic residues in the ferrichrome binding protein, FhuD2. Taken together, these results support the hypothesis that Glu-Arg salt bridges are critical for the interaction of the ligand binding protein with the transmembrane domains FhuB and FhuG. This hypothesis was further studied by "charge swapping" experiments whereby we constructed a S. aureus strain expressing FhuD2 with conserved residues Glu-97 and Glu-231 replaced by Arg and FhuB and FhuG with conserved basic residues Arg-71 and Arg-61, respectively, replaced by Glu. A strain containing this combination of substitutions restored partial function to the ferrichrome transporter. The results provide a direct demonstration of the functional importance of conserved basic residues on the extracellular surface of the ferrichrome permease in the Gram-positive bacterium S. aureus.
authors.affiliation: Department of Microbiology and Immunology, University of Western Ontario, London, ON, N6A 5C1, Canada.
authors.forename: Enrique D
authors.initials: ED
authors.lastname: Vinés
authors.forename: Craig D
authors.initials: CD
authors.lastname: Speziali
authors.forename: David E
authors.initials: DE
authors.lastname: Heinrichs
mesh_terms: D001692:Biological Transport; D004151:Dipeptides / Q000378:metabolism*; D005291:Ferrichrome / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008958:Models, Molecular; D013211:Staphylococcus aureus / Q000254:growth & development / Q000378:metabolism*
chemical_list: D004151:Dipeptides; D026901:Membrane Transport Proteins; D005291:Ferrichrome; C024362:arginine glutamate