Institute of Molecular Biology and Biophysics, Department of Biology, ETH Zurich, Switzerland.
Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland.
Nature. 2018 Nov;563(7731):426-430. doi: 10.1038/s41586-018-0680-3. Epub 2018 Nov 7.
ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, blood-testis and maternal-fetal barriers. Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs. Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies. However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (ES) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of ES can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a 'plug' between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude ES and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors.
ABCG2 是一种 ATP 结合盒（ABC）家族的转运蛋白，在各种组织和组织屏障的细胞膜中表达，包括血脑、血睾和母婴屏障。在 ATP 的驱动下，它转运内源性底物，影响许多药物的药代动力学，并防止广泛的外源性化学物质，包括抗癌药物的侵害。先前的研究揭示了 ABCG2 的结构及其被小分子和抗体抑制的结构基础。然而，底物识别和 ATP 驱动的转运机制尚不清楚。在这里，我们展示了人源 ABCG2 在底物结合的前移位状态和 ATP 结合的后移位状态下的高分辨率冷冻电镜（cryo-EM）结构。对于这两种结构，我们使用了一个突变体，其中一个谷氨酸被谷氨酰胺取代（ABCG2），这导致 ATP 酶和转运速率降低，并促进了结构研究的构象捕获。在底物结合状态下，单分子雌酮-3-硫酸盐（ES）结合在膜的中央、疏水性和细胞质面向的腔中，大约在膜的一半处。在观察到的结合模式中，只有一个 ES 分子可以结合。在 ATP 结合状态下，底物结合腔已经塌陷，而外部腔已经打开到膜的细胞外侧面。ATP 诱导的构象变化包括跨膜域的刚性体位移、核苷酸结合域（NBD）的枢轴转动以及 NBD 亚域的相对取向的变化。转运和 ATP 酶活性的突变和体外表征表明，特定残基在底物识别中的作用，包括形成两个腔之间“塞子”的亮氨酸残基。我们的结果显示了 ABCG2 如何利用 ATP 结合的能量来排出 ES 和其他底物，并表明化合物的大小和结合亲和力对于区分底物和抑制剂很重要。
