Structures of a Na+-coupled, substrate-bound MATE multidrug transporter
Structures of a Na+-coupled, substrate-bound MATE multidrug transporter
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DOI：
10.1073/pnas.1219901110
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发表时间：
2013-02-05
影响因子：
11.1
通讯作者：
Koide, Shohei
中科院分区：
文献类型：
--
作者：
Lu, Min;Symersky, Jindrich;Koide, Shohei
属于多药和毒性化合物排出（MATE）家族的多药转运蛋白通过消散预先存在的Na+或H+梯度来排出不同的亲脂性和阳离子药物穿过细胞膜。尽管其临床意义，MATE蛋白的转运机制仍然知之甚少，主要是由于缺乏对底物结合转运蛋白的结构信息。在这里，我们报告的Na+耦合MATE转运NorM淋球菌在复合物与三个不同的易位基板（乙锭，罗丹明6 G，和tetraphenylphosphonium），以及Cs+（Na+同源物），所有捕获在细胞外面向和药物结合状态的晶体结构。这些结构揭示了一个多药物结合腔，其中充满了四个带负电荷的氨基酸和令人惊讶的有限的疏水性部分，这与芳香族氨基酸在多药物识别中发挥重要作用的普遍信念形成鲜明对比。此外，我们发现了一种不常见的阳离子-p相互作用的Na+-结合位点位于药物结合腔外，并验证了生物相关性的底物和阳离子结合位点进行耐药性和运输试验。此外，我们发现潜在的重排后，钠+诱导的药物出口至少有两个跨膜螺旋。基于我们的结构和功能分析，我们建议，Na+触发多药挤出诱导蛋白质构象的变化，而不是通过直接竞争的底物结合氨基酸。这种情况与典型的反向转运机制不同，在反向转运机制中，底物和配偶体都竞争转运蛋白中的共享结合位点。总的来说，我们的研究结果为详细了解多药转运机制迈出了重要的一步。
Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na+ or H+ gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na+-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs+ (a Na+ congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-p interaction in the Na+-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate-and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na+-induced drug export. Based on our structural and functional analyses, we suggest that Na+ triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.