Department of Chemistry, University of Toronto, Toronto, ON M5S 3H6, Canada.
Department of Chemical and Physical Sciences, University of Toronto, Mississauga, ON L5L 1C6, Canada.
Proc Natl Acad Sci U S A. 2021 May 25;118(21). doi: 10.1073/pnas.2025853118.
ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.
ATP 结合盒（ABC）蛋白通常在溶质的跨膜主动运输中发挥作用。ABC 核心结构由两个跨膜结构域（TMD1 和 TMD2）和两个胞质核苷酸结合结构域（NBD1 和 NBD2）组成。ABC 家族的 C 亚家族（ABCC）的一些成员，包括人类多药耐药蛋白（MRP），还具有一个 N 端跨膜结构域（TMD0），该结构域包含五个跨膜α螺旋，并通过 L0 接头与 ABC 核心相连。虽然 SUR1 解析了 TMD0，这是异源八聚体 ATP 敏感性 K 通道的一部分，但关于单体 ABC 转运蛋白中 TMD0 的结构知之甚少。在这里，我们通过电子 cryo 显微镜（cryo-EM）确定了酵母镉因子 1 蛋白（Ycf1p）的结构，该蛋白是人类 MRP1 的同源物。将 Ycf1p、SUR1 与一个 TMD0 分辨率较低的 MRP1 结构进行比较表明，TMD0 相对于 ABC 核心可以采用不同的取向，包括 Ycf1p 和 SUR1 之间约 145°的旋转。cryo-EM 图谱还揭示了连接 NBD1 和 TMD2 的调节（R）区的片段与 L0 接头、NBD1 和 TMD2 相互作用。这些相互作用，结合与无 R 区的 NBD1 进行的荧光猝灭实验，表明 R 区的翻译后修饰如何调节 ABC 蛋白的活性。将来自 MRP2 和 MRP6 的已知突变映射到 Ycf1p 结构上，解释了这些蛋白中涉及 TMD0 和 R 区的突变如何导致疾病。
