Functional characterization of Escherichia coli MsbA -: Interaction with nucleotides and substrates
Functional characterization of Escherichia coli MsbA -: Interaction with nucleotides and substrates
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DOI：
10.1074/jbc.m708274200
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发表时间：
2008-05-09
影响因子：
4.8
通讯作者：
Sharom, Frances J.
中科院分区：
文献类型：
--
作者：
Eckford, Paul D. W.;Sharom, Frances J.
大肠杆菌MsbA蛋白是ATP结合盒超家族的65-kDa成员。它被认为是一种ATP依赖性脂质移位酶，将脂质A从细胞质膜的内小叶转运到外小叶。具有高ATP酶活性的MsbA被分离，并发现在洗涤剂溶液中是同源二聚体。蛋白ATP酶活性被钒酸盐抑制，并表现出不同的刺激和抑制模式的脂质A和其他化合物。用丙烯酰胺动态猝灭法研究了该蛋白的色氨酸荧光特性，结果表明该蛋白与类脂A结合后发生了构象变化。荧光猝灭被用来表征MsbA与核苷酸和各种推定的底物，包括脂质，脂质样化合物和药物的相互作用。MsbA对ATP具有类似于2 mM的表观结合亲和力，并且还结合不可水解的ATP类似物和荧光ATP衍生物。假定的底物脂质A与蛋白质相互作用的亲和力为6.4 μ M。已知作为ABC多药转运蛋白底物的药物也与MsbA相互作用，亲和力在0.25-50 μ M范围内。这项研究代表了第一次使用荧光的方法来估计MsbA结合亲和力的核苷酸和推定的运输底物。
The Escherichia coli MsbA protein is a 65-kDa member of the ATP-binding cassette superfamily. It is thought to function as an ATP-dependent lipid translocase that transports lipid A from the inner to the outer leaflet of the cytoplasmic membrane. MsbA with high ATPase activity was isolated and found to be homodimeric in detergent solution. The protein ATPase activity was inhibited by vanadate and showed variable patterns of stimulation and inhibition by lipid A and other compounds. The intrinsic tryptophan fluorescence of the protein was characterized, and dynamic quenching using acrylamide showed that a conformational change took place on binding of lipid A. Fluorescence quenching was used to characterize the interactions of MsbA with nucleotides and various putative substrates, including lipids, lipid-like compounds, and drugs. MsbA had an apparent binding affinity for ATP of similar to 2 mM and also bound nonhydrolyzable ATP analogs and fluorescent ATP derivatives. The putative substrate lipid A interacted with the protein with an affinity of 6.4 mu M. Drugs that are known to be substrates for ABC multidrug transporters also interacted with MsbA with affinities in the range 0.25-50 mu M. This study represents the first use of fluorescence approaches to estimate MsbA binding affinities for nucleotides and putative transport substrates.