Nucleotide dependent monomer/dimer equilibrium of OpuAA, the nucleotide-binding protein of the osmotically regulated ABC transporter OpuA from Bacillus subtilis

被引:43
作者
Horn, C
Bremer, E
Schmitt, L
机构
[1] Goethe Univ Frankfurt, Inst Biochem, Bioctr N210, D-60439 Frankfurt, Germany
[2] Univ Marburg, Microbiol Lab, Dept Biol, D-35032 Marburg, Germany
关键词
ABC transporter; nucleotide-binding protein; ATPase; monomer/dimer equilibrium; TNP-ATP;
D O I
10.1016/j.jmb.2003.09.079
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 over-expressed 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 muM. Under high ionic strength assay conditions, the monomer/dimer inter-conversion 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/Mg2+), 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/Mg2+ 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. (C) 2003 Elsevier Ltd. All rights reserved.
引用
收藏
页码:403 / 419
页数:17
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