Structure and drug resistance of the Plasmodium falciparum transporter PfCRT
Structure and drug resistance of the Plasmodium falciparum transporter PfCRT
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DOI：
10.1038/s41586-019-1795-x
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发表时间：
2019-12-12
期刊：
影响因子：
64.8
通讯作者：
Mancia, Filippo
中科院分区：
文献类型：
--
作者：
Kim, Jonathan;Tan, Yong Zi;Mancia, Filippo
抗药性恶性疟原虫的出现和传播阻碍了全球控制和消除疟疾的努力。几十年来，疟疾的治疗一直依赖于氯喹（CQ），这是一种安全且负担得起的4-氨基喹啉，对红细胞内无性血液阶段寄生虫非常有效，直到东南亚和南美洲出现耐药性并在全球蔓延（1）。化学相关的当前一线复方药物哌喹（PPQ）的临床耐药性现已出现区域性，降低了其疗效（2）。对CQ和PPQ的耐药性与恶性疟原虫CQ耐药转运蛋白PfCRT中不同的点突变组相关，PfCRT是药物/代谢物转运蛋白超家族的一个49 kDa成员，穿过寄生虫酸性消化空泡的膜（3-9）。在这里，我们提出的结构，在3.2埃的分辨率，PfCRT异构体的CQ耐药，PPQ敏感的南美7 G8寄生虫，使用单粒子冷冻电子显微镜和抗原结合片段技术。导致CQ和PPQ抗性的突变主要定位于不同螺旋上的中度保守位点，这些位点排列在中心带负电荷的空腔内，表明该空腔是与带正电荷的CQ和PPQ相互作用的主要位点。结合和转运研究表明，7 G8同种型以相当的亲和力结合两种药物，并且这些药物是相互竞争的。7 G8亚型以膜电位和pH依赖性方式转运CQ，这与驱动CQ抗性的主动外排机制一致（5），但不转运PPQ。对新出现的PfCRT F145 I和C350 R突变（分别与亚洲和南美洲的PPQ敏感性降低相关（6，9））的功能研究表明，它们能够介导7 G8变体蛋白中的PPQ转运，并赋予基因编辑的寄生虫耐药性。结构、功能和计算机模拟分析表明，不同的机制特征介导PfCRT变体对CQ和PPQ的耐药性。这些数据提供了原子水平的见解，抗疟治疗失败的这一关键介质的分子机制。
The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide(1). Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy(2). Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite(3-9). Here we present the structure, at 3.2 angstrom resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance(5), but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively(6,9), reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.