ABCG2是一种ATP结合盒转运蛋白，可以输出多种异物化合物，并且已被认为是癌细胞多药耐药的一个影响因素。对ABCG2的底物和抑制剂相互作用进行了广泛研究，并开发了小分子抑制剂，以防止抗癌药物从肿瘤细胞中输出。在这里，我们探索了除了ABCG2底物结合口袋以外的其他靶点的抑制剂的潜力。我们开发了针对ABCG2的新型纳米抗体，并使用功能分析选择三种抑制性纳米抗体（Nb8、Nb17和Nb96）进行单颗粒冷冻电镜结构研究。我们的研究结果表明，这些纳米抗体与核苷酸结合结构域的不同区域发生非同源性结合。两个Nb8与NBD的顶端结合，并阻止了其完全闭合。Nb17与转运蛋白的二重轴附近结合，并与两个NBD相互作用。Nb96与NBD的侧面结合，固定了与ATP结合和水解有关的关键模体连接的区域。这三种纳米抗体都阻止了转运蛋白发生所需的构象变化。这些发现推进了我们对外源结合物调节ABCG2的分子基础的理解，这可能有助于开发新一代抑制剂。此外，这是首个使用纳米抗体调节人类多药耐药转运蛋白的例子。版权所有©2023作者。由Elsevier Ltd.出版。保留所有权利。

ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.