Residues contributing to drug transport by ABCG2 are localised to multiple drug-binding pockets.
Residues contributing to drug transport by ABCG2 are localised to multiple drug-binding pockets.
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DOI：
10.1042/bcj20170923
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发表时间：
2018-05-04
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影响因子：
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通讯作者：
Kerr ID
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作者：
Cox MH;Kapoor P;Briggs DA;Kerr ID
atp结合盒转运体ABCG2的多药结合和转运是白血病临床化疗耐药的一个因素，也是许多其他处方药的药代动力学特征的一个促进因素。尽管它很重要，但多药转运的结构基础，即转运多种不同化学物质的能力，仍然难以捉摸。先前的研究表明，至少有两个位于转运体跨膜螺旋3 （TM3）细胞质端的残基在药物转运中起作用。我们假设其他残基，无论是在TM3的纵向跨度上，还是在其他TM螺旋的细胞内末端的垂直切片上，也有助于ABCG2的药物结合和运输。在哺乳动物稳定细胞系表达系统中，在~ 30个位置制备ABCG2单点突变异构体，分析其对蛋白质表达、定位（western blotting，共聚焦显微镜）和功能（流式细胞术）的影响。我们的数据是根据ABCG蛋白亚家族的最新结构数据进行解释的，这使我们能够提出药物米托蒽醌（MX）的表面结合位点以及同一药物的第二个埋藏位点。对空间上分隔这两个位点的残基的进一步突变分析提示我们ABCG2转运MX的分子和结构途径。
Multidrug binding and transport by the ATP-binding cassette transporter ABCG2 is a factor in the clinical resistance to chemotherapy in leukaemia, and a contributory factor to the pharmacokinetic profiles of many other prescribed drugs. Despite its importance, the structural basis of multidrug transport, i.e. the ability to transport multiple distinct chemicals, has remained elusive. Previous research has shown that at least two residues positioned towards the cytoplasmic end of transmembrane helix 3 (TM3) of the transporter play a role in drug transport. We hypothesised that other residues, either in the longitudinal span of TM3, or a perpendicular slice through the intracellular end of other TM helices would also contribute to drug binding and transport by ABCG2. Single-point mutant isoforms of ABCG2 were made at ∼30 positions and were analysed for effects on protein expression, localisation (western blotting, confocal microscopy) and function (flow cytometry) in a mammalian stable cell line expression system. Our data were interpreted in terms of recent structural data on the ABCG protein subfamily and enabled us to propose a surface-binding site for the drug mitoxantrone (MX) as well as a second, buried site for the same drug. Further mutational analysis of residues that spatially separate these two sites prompts us to suggest a molecular and structural pathway for MX transport by ABCG2.