The multidrug resistance protein MRP1 is an ATP-binding cassette (ABC) transporter that confers resistance to many anticancer drugs and plays a role in the disposition and efficacy of several opiates, antidepressants, statins, and antibiotics. In addition, MRP1 regulates redox homeostasis, inflammation, and hormone secretion. Using electron cryomicroscopy, we determined the molecular structures of bovine MRP1 in two conformations: an apo form at 3.5 Å without any added substrate and a complex form at 3.3 Å with one of its physiological substrates, leukotriene C. These structures show that by forming a single bipartite binding site, MRP1 can recognize a spectrum of substrates with different chemical structures. We also observed large conformational changes induced by leukotriene C, explaining how substrate binding primes the transporter for ATP hydrolysis. Structural comparison of MRP1 and P-glycoprotein advances our understanding of the common and unique properties of these two important molecules in multidrug resistance to chemotherapy.

The multidrug resistance protein MRP1 is an ATP-driven pump that confers resistance to chemotherapy. Previously, we have shown that intracellular substrates are recruited to a bipartite binding site when the transporter rests in an inward-facing conformation. A key question remains: how are high-affinity substrates transferred across the membrane and released outside the cell? Using electron cryomicroscopy, we show here that ATP binding opens the transport pathway to the extracellular space and reconfigures the substrate-binding site such that it relinquishes its affinity for substrate. Thus, substrate is released prior to ATP hydrolysis. With this result, we now have a complete description of the conformational cycle that enables substrate transfer in a eukaryotic ABC exporter.

SummaryNucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine plays a role in a variety of signaling processes 1,2 . Transporting nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signaling. Due to their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes [3][4][5] . A wide range of nucleoside-derived drugs has been shown to depend, at least in part, on NTs for transport across cell membranes including anticancer drugs (e.g., Ara-C and gemcitabine) and antiviral drugs (e.g., AZT and ribavirin) 4,[6][7][8][9][10][11][12][13] . Concentrative nucleoside transporters (CNTs), members of the solute carrier transporter superfamily SLC28, use an ion gradient to actively transport nucleosides as well as nucleosidederived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by CNTs is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 Å. Our functional data show that the transporter utilizes a sodium gradient for nucleoside transport like its human orthologs. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.Humans have three isoforms of CNT and the substrate specificity and tissue distribution are different among the isoforms [14][15][16] . Knowledge of the mechanism of these transporters would help us not only to understand physiological processes associated with nucleosides but also to provide a framework for future drug design to improve nucleoside drug delivery.Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms * Correspondence and requests for materials should be addressed to S.-Y. Lee. sylee@biochem.duke.edu, tel: 919-684-1005, fax: 919-613-5145. Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions Z.J. expressed, purified, and crystallized vcCNT. Z.J. performed radioactive flux and cross-linking experiments. C.-G.C. participated in part of the vcCNT crystallization and generated mutants for crystallization and functional studies. Z.J. and S.-Y. L. collected and processed the data, solved the structure, and wrote the paper. S.-Y.L designed the study. All authors discussed the results and commented on the manuscript.Atomic coord...