Nucleotide dependent monomer/dimer equilibrium of OpuAA, the nucleotide-binding protein of the osmotically regulated ABC transporter OpuA from Bacillus subtilis.
The OpuA system of Bacillus subtilis is a member of the substrate-binding-protein-dependent ABC transporter superfamily and serves for the uptake of the compatible solute glycine betaine under hyperosmotic growth conditions. Here, we have characterized the nucleotide-binding protein (OpuAA) of the B.subtilis OpuA transporter in vitro. OpuAA was overexpressed heterologously in Escherichia coli as a hexahistidine tag fusion protein and purified to homogeneity by affinity and size exclusion chromatography (SEC). Dynamic monomer/dimer equilibrium was observed for OpuAA, and the K(D) value was determined to be 6 microM. Under high ionic strength assay conditions, the monomer/dimer interconversion was diminished, which enabled separation of both species by SEC and separate analysis of both monomeric and dimeric OpuAA. In the presence of 1 M NaCl, monomeric OpuAA showed a basal ATPase activity (K(M)=0.45 mM; k(2)=2.3 min(-1)), whereas dimeric OpuAA showed little ATPase activity under this condition. The addition of nucleotides influenced the monomer/dimer ratio of OpuAA, demonstrating different oligomeric states during its catalytic cycle. The monomer was the preferred species under post-hydrolysis conditions (e.g. ADP/Mg(2+)), whereas the dimer dominated the nucleotide-free and ATP-bound states. The affinity and stoichiometry of monomeric or dimeric OpuAA/ATP complexes were determined by means of the fluorescent ATP-analog TNP-ATP. One molecule of TNP-ATP was bound in the monomeric state and two TNP-ATP molecules were detected in the dimeric state of OpuAA. Binding of TNP-ADP/Mg(2+) to dimeric OpuAA induced a conformational change that led to the decay of the dimer. On the basis of our data, we propose a model that couples changes in the oligomeric state of OpuAA with ATP hydrolysis.
Horn
Carsten
C
Institute of Biochemistry, Biocenter N210, Johann-Wolfgang Goethe University Frankfurt, Marie-Curie Str. 9, 60439, Frankfurt, Germany.
Bremer
Erhard
E
Schmitt
Lutz
L
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001622:Betaine / Q000378:metabolism; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D008074:Lipoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011108:Polymers; D014882:Water-Electrolyte Balance
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008074:Lipoproteins; C108613:OpuAC protein, Bacillus subtilis; D011108:Polymers; D001622:Betaine; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases