Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis
Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis
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DOI：
10.1006/jmbi.2001.5309
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发表时间：
2002-02-01
影响因子：
5.6
通讯作者：
Marco, S
中科院分区：
文献类型：
--
作者：
Chami, M;Steinfels, E;Marco, S
YvcC是枯草芽孢杆菌的多药转运蛋白，是三磷酸腺苷结合盒超家族的成员之一，与人多药耐药P-糖蛋白的每一半高度同源，也与其他几种细菌半ABC转运蛋白高度同源。在这里，纯化的重组组氨酸标记的YvcC被重组为脂质双层。从YvcC-脂类胶束中受控和部分去除洗涤剂可以产生特别有趣的脂类-洗涤剂-YvcC环状颗粒，直径约40 nm，非常适合于冷冻电子显微镜下的单颗粒分析。此外，这些组氨酸标记的环状颗粒结合到由Ni2+螯合头部基团功能化的脂层上，产生了优先的垂直取向，消除了最终三维重建中缺失的锥体。从这样的分析中，已经确定了2.5 nm分辨率的计算体积，从而详细地洞察了这种半ABC转运体在膜内的结构组织。环状颗粒中的重复单元与YvcC的同源二聚体结构一致。每个亚基由3个结构域组成：一个5行高的跨膜区、一个高约4 nm、直径约2 nm的柄和一个直径约5-6 nm的细胞质小叶。最新的结构域与已报道的Hisp的X射线结构相吻合，被鉴定为核苷酸结合结构域(NBD)。YvcC同源二聚体的三维重建与最新的来自大肠杆菌的MSBA同源二聚体的X射线结晶学数据进行了很好的比较，支持组成同源二聚体的两个亚基之间存在一个中心开口室。此外，包埋在膜中的YvcC的3D重建揭示了同源二聚体中两个NBDS位点的不对称组织，以及两个同源二聚体之间的二聚体相互作用。(C)2001年爱思唯尔科学有限公司。
YvcC, a multidrug transporter from Bacillus subtilis, is a member of the ATP-binding cassette superfamily, highly homologous to each half of human multidrug-resistance P-glycoprotein and to several other bacterial half-ABC transporters. Here, the purified recombinant histidine-tagged YvcC has been reconstituted into a lipid bilayer. Controlled and partial detergent removal from YvcC-lipid micelles allowed the production of particularly interesting lipid-detergent-YvcC ring-shaped particles, about 40 nm in diameter, well suited for single particle analysis by cryo-electron microscopy. Furthermore, binding of these histidine-tagged ring-shaped particles to lipid layers functionalized with a Ni2+-chelating head group generated a preferential perpendicular orientation, eliminating the missing cone in the final three-dimensional reconstruction. From such analysis, a computed volume has been determined to 2.5 nm resolution giving a detailed insight into the structural organization of this half-ABC transporter within a membrane. The repetitive unit in the ring-shaped particles is consistent with a homodimeric organization of YvcC. Each subunit was composed of three domains: a 5 run height transmembrane region, a stalk of about 4 nm in height and 2 nm in diameter, and a cytoplasmic lobe of about 5-6 nm in diameter. The latest domain, which fitted with the reported X-ray structure of HisP, was identified as the nucleotide-binding domain (NBD). The 3D reconstruction of the YvcC homodimer well compared with the very recent X-ray crystallographic data on the MsbA homodimer from Escherichia coli, supporting the existence of a central open chamber between the two subunits constituting the homodimer. In addition, the 3D reconstruction of YvcC embedded in a membrane revealed an asymmetric organization of the two NBDs sites within the homodimer, as well as a dimeric interaction between two homodimers. (C) 2001 Elsevier Science Ltd.