Abstract
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.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Structure |
| Vol/bind | 31 |
| Udgave nummer | 11 |
| Sider (fra-til) | 1407-1418.e6 |
| ISSN | 0969-2126 |
| DOI | |
| Status | Udgivet - 2023 |
Bibliografisk note
Funding Information:The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant NNF14CC0001 ). This work was also supported by an NNF Hallas-Møller Emerging Investigator ( NNF17OC0031006 ). N.M.I.T. is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen. M.B. acknowledges the Neye Foundation for support. We thank the Danish Cryo-EM Facility at the Core Facility for Integrated Microscopy (CFIM) at the University of Copenhagen. Part of the data processing was performed at the Computerome, the Danish National Computer for Life Sciences.
Funding Information:
The Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant NNF14CC0001). This work was also supported by an NNF Hallas-Møller Emerging Investigator (NNF17OC0031006). N.M.I.T. is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of Copenhagen. M.B. acknowledges the Neye Foundation for support. We thank the Danish Cryo-EM Facility at the Core Facility for Integrated Microscopy (CFIM) at the University of Copenhagen. Part of the data processing was performed at the Computerome, the Danish National Computer for Life Sciences. We thank Katharina Duerr (University of Oxford) for kindly supplying us with a vial of the purified pHTBV1.1 vector, Jonathan Elegheert (IINS) for kindly supplying us with the expression plasmid for the HRV3C protease (as well as protocols for expression and purification), and Kenneth A. Matreyek (Case Western Reserve University) for kindly supplying vials of the parental HEK293T LLP-Int-Blast cell line, as well as protocols for maintaining and establishing derivative cell lines. M.B. and N.M.I.T. conceptualized the research project and planned the experiments. M.B. carried out all expressions, purifications, and sample preparations for electron microscopy and ATPase activity assays and analyzed the data. T.P. and M.B. collected and analyzed the electron microscopy data. I.R. and M.B. built, refined, and validated the structural models. M.B. conducted the cellular drug susceptibility assays and analyzed the data. I.R. and M.B. carried out all molecular cloning and generated the derivative stable cell lines. M.B. wrote the first draft of the paper and prepared all the figures together with N.M.I.T. All authors contributed to the revision of the manuscript. The authors declare no competing interests. We support inclusive, diverse, and equitable conduct of research.
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