The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites.
The ABC transporter MsbA interacts with lipid A and amphipathic drugs at different sites.
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DOI：
10.1042/bj20081364
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发表时间：
2009-04-15
期刊：
影响因子：
--
通讯作者：
Sharom FJ
中科院分区：
文献类型：
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作者：
Siarheyeva A;Sharom FJ
MsbA是一种必需的ABC（ATP结合盒）转运蛋白，参与脂多糖A在革兰氏阴性菌细胞质膜上的转运。该蛋白还与两亲性药物的外排有关。纯化的野生型MsbA在C315位点被荧光探针MIANS[2 -（4′ - 马来酰亚胺基苯胺基）萘 - 6 - 磺酸]按化学计量标记，C315位于连接跨膜螺旋6和核苷酸结合结构域的胞内结构域内。MsbA - MIANS显示出高ATP酶活性，其折叠和稳定性未发生改变。在两亲性药物存在的情况下，MIANS标记MsbA的初始速率降低，这表明这些化合物的结合改变了蛋白质的构象。MsbA - MIANS的荧光可被核苷酸、脂多糖A和各种药物饱和猝灭，结合的Kd值估计在0.35 - 10 μM范围内。脂多糖A和柔红霉素能够同时与MsbA - MIANS结合，这意味着它们占据不同的结合位点。核苷酸与脂多糖A/柔红霉素结合的效应是累加的，且结合无顺序。当柔红霉素结合位点首先被占据时，MsbA结合脂多糖A的Kd值显著降低，并且核苷酸的预先结合也调节了脂多糖A的结合亲和力。这些结果表明，MsbA包含两个底物结合位点，它们与核苷酸结合结构域相互作用且彼此之间也相互作用。一个是生理底物脂多糖A的高亲和力结合位点，另一个位点以相当的亲和力与药物相互作用。因此，MsbA可能兼具脂类翻转酶和多药转运蛋白的功能。
MsbA is an essential ABC (ATP-binding cassette) transporter involved in lipid A transport across the cytoplasmic membrane of Gram-negative bacteria. The protein has also been linked to efflux of amphipathic drugs. Purified wild-type MsbA was labelled stoichiometrically with the fluorescent probe MIANS [2-(4′-maleimidylanilino)naphthalene-6-sulfonic acid] on C315, which is located within the intracellular domain connecting transmembrane helix 6 and the nucleotide-binding domain. MsbA–MIANS displayed high ATPase activity, and its folding and stability were unchanged. The initial rate of MsbA labelling by MIANS was reduced in the presence of amphipathic drugs, suggesting that binding of these compounds alters the protein conformation. The fluorescence of MsbA–MIANS was saturably quenched by nucleotides, lipid A and various drugs, and estimates of the Kd values for binding fell in the range of 0.35–10 μM. Lipid A and daunorubicin were able to bind to MsbA–MIANS simultaneously, implying that they occupy different binding sites. The effects of nucleotide and lipid A/daunorubicin binding were additive, and binding was not ordered. The Kd of MsbA for binding lipid A was substantially decreased when the daunorubicin binding site was occupied first, and prior binding of nucleotide also modulated lipid A binding affinity. These results indicate that MsbA contains two substrate-binding sites that communicate with both the nucleotide-binding domain and with each other. One is a high affinity binding site for the physiological substrate, lipid A, and the other site interacts with drugs with comparable affinity. Thus MsbA may function as both a lipid flippase and a multidrug transporter.