Three-dimensional reconstruction of the Saccharomyces cerevisiae multidrug resistance protein Pdr5p
Three-dimensional reconstruction of the Saccharomyces cerevisiae multidrug resistance protein Pdr5p
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DOI：
10.1074/jbc.m212198200
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发表时间：
2003-04-04
影响因子：
4.8
通讯作者：
Rigaud, JL
中科院分区：
文献类型：
--
作者：
Ferreira-Pereira, A;Marco, S;Rigaud, JL
Pdr5p 是酿酒酵母中主要的多药输出蛋白，是 ATP 结合盒 (ABC) 超家族的成员。尽管跨膜和 ATP 结合域的拓扑结构颠倒，但 Pdr5p 与人类 MDR1-Pgp 具有相似的底物识别和转运机制。六组氨酸标记的 Pdr5p 多药转运蛋白在其他 ABC 基因已被删除的酵母菌株中高度过表达。经过溶解和纯化后，160 kDa 重组 Pdr5p 已重构为脂质双层。从 Pdr5p-脂质去污剂胶束中控制去污剂去除可以产生与脂质体和蛋白脂质体共存的特殊方形颗粒。这些边长为 11 nm 的颗粒非常适合通过电子显微镜进行单颗粒分析。通过此类分析，计算体积已确定为 25 埃分辨率，从而深入了解 Pdr5p 的结构组织。与报道的不同细菌 ABC 转运蛋白的结构进行比较，与方形颗粒中 Pdr5p 的二聚体组织一致。每个单体由三个子区域组成，对应于高度约50埃的膜区域，该膜区域连接两个高度分开的约40埃的突出茎，每个子区域以直径约50-60埃的细胞质核苷酸结合结构域（NBD）叶结束。 Pdr5p 的三维重建揭示了两个 NBD 的紧密排列和结构不对称组织，它们在单体内垂直取向。 NBD 相对于茎存在不同的角度位置，表明催化循环期间存在旋转运动。
Pdr5p, the major multidrug exporter in Saccharomyces cerevisiae, is a member of the ATP-binding cassette (ABC) superfamily. Pdr5p shares similar mechanisms of substrate recognition and transport with the human MDR1-Pgp, despite an inverted topology of transmembrane and ATP-binding domains. The hexahistidine-tagged Pdr5p multidrug transporter was highly overexpressed in yeast strains where other ABC genes have been deleted. After solubilization and purification, the 160-kDa recombinant Pdr5p has been reconstituted into a lipid bilayer. Controlled detergent removal from Pdr5p-lipid-detergent micelles allowed the production of peculiar square-shaped particles coexisting with liposomes and proteoliposomes. These particles having 11 nm in side were well suited for single particle analysis by electron microscopy. From such analysis, a computed volume has been determined at 25-Angstrom resolution, giving insight into the structural organization of Pdr5p. Comparison with the reported structures of different bacterial ABC transporters was consistent with a dimeric organization of Pdr5p in the square particles. Each monomer was composed of three subregions corresponding to a membrane region of about 50 Angstrom in height that joins two well separated protruding stalks of about 40 Angstrom in height, ending each one with a cytoplasmic nucleotide-binding domain (NBD) lobe of about 50-60 Angstrom in diameter. The three-dimensional reconstruction of Pdr5p revealed a close arrangement and a structural asymmetric organization of the two NBDs that appeared oriented perpendicularly within a monomer. The existence of different angular positions of the NBDs, with respect to the stalks, suggest rotational movements during the catalytic cycle.