多药耐药相关蛋白4（MRP4）是一种在多个组织屏障中表达的ATP结合盒（ABC）转运蛋白，能主动排出各种药物化合物。MRP4的过度表达导致对临床使用的抗肿瘤药物产生耐药性，使其成为对抗多药耐药性的高度吸引的治疗靶点。在这里，我们报道了多种与生理相关状态的冻结电子显微镜（cryo-EM）结构，这些结构包括嵌入脂质双分子层的人类MRP4与两种广泛使用的化疗药物以及与其天然底物之间的复合物。这些结构显示了这些化学多样的底物的配位上的明显相似性和独特差异，并结合功能和突变分析，揭示了运输机制的分子细节。我们的研究为MRP4异常广泛的底物特异性提供了关键的见解，并对多药转运蛋白的一般理解做出了重要贡献。版权所有© 2023 作者。由Elsevier Ltd.出版。保留所有权利。

Multidrug resistance-associated protein 4 (MRP4) is an ATP-binding cassette (ABC) transporter expressed at multiple tissue barriers where it actively extrudes a wide variety of drug compounds. Overexpression of MRP4 provides resistance to clinically used antineoplastic agents, making it a highly attractive therapeutic target for countering multidrug resistance. Here, we report cryo-EM structures of multiple physiologically relevant states of lipid bilayer-embedded human MRP4, including complexes between MRP4 and two widely used chemotherapeutic agents and a complex between MRP4 and its native substrate. The structures display clear similarities and distinct differences in the coordination of these chemically diverse substrates and, in combination with functional and mutational analysis, reveal molecular details of the transport mechanism. Our study provides key insights into the unusually broad substrate specificity of MRP4 and constitutes an important contribution toward a general understanding of multidrug transporters.Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.