Interaction between ATP, oleandomycin and the OleB ATP-binding cassette transporter of Streptomyces antibioticus involved in oleandomycin secretion.
The OleB protein of Streptomyces antibioticus, oleandomycin (OM) producer, constitutes an ATP-binding cassette transporter containing two nucleotide-binding domains and is involved in OM resistance and its secretion in this producer strain. We have characterized some properties of the first nucleotide-binding domain of OleB using an overexpressed fusion protein (MBP-OleB') between a maltose-binding protein (MBP) and the first half of OleB (OleB'). Extrinsic fluorescence of the base-modified fluorescent nucleotide analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon ATP) and 2'(3')-o-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate was determined in the presence of MBP and the fusion protein MBP-OleB', and it was found that epsilon ATP binds to MBP-OleB' with a stoichiometry of 0.9. Measurements of the intrinsic fluorescence of the MBP-OleB' fusion protein indicated that ATP induces a decrease in the accessibility of the MBP-OleB' tryptophans to acrylamide, an indication of a folding effect. This conclusion was confirmed by the fact that ATP also induces considerable stabilization against guanidine chloride denaturation of MBP-OleB'. Two effects were found to be associated with the presence of Mg2+ ions: (1) an increase in the quenching of MBP-OleB' intrinsic fluorescence by ATP; and (2) an increase in the accessibility of MBP-OleB' tryptophans to acrylamide. Significant changes in the intrinsic fluorescence of the fusion protein were also observed in the presence of OM, demonstrating the existence of interaction between the transporter and the antibiotic in the absence of any hydrophobic membrane component.
Buche
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A
Departamento de Biología Funcional, Universidad de Oviedo, Spain.
Méndez
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C
Salas
J A
JA
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D020106:Acrylamide; D000178:Acrylamides / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000378:metabolism; D004917:Erythromycin / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D058619:Maltose-Binding Proteins; D009827:Oleandomycin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013303:Streptomyces antibioticus
D018528:ATP-Binding Cassette Transporters; D000178:Acrylamides; D001426:Bacterial Proteins; D002352:Carrier Proteins; D006146:Guanidines; D058619:Maltose-Binding Proteins; C095776:OleB protein, Streptomyces antibioticus; D020106:Acrylamide; D004917:Erythromycin; D000255:Adenosine Triphosphate; D019791:Guanidine; D009827:Oleandomycin