The substrate binding site in MRP1 contains a positively charged region and a hydrophobic region.
Major finding: The substrate binding site in MRP1 contains a positively charged region and a hydrophobic region.
Approach: Electron cryomicroscopy was performed to resolve the structures of MRP1 alone and in complex.
Impact: The structurally plastic substrate binding site in MRP1 enables the recognition of diverse molecules.
ATP binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1) and P-glycoprotein (P-gp), utilize ATP hydrolysis to transport drugs out of cancer cells and thus promote chemoresistance. However, whereas P-gp has the archetypical structure of an ABC transporter and recognizes hydrophobic substrates, MRP1 has a noncanonical structure—an additional N-terminal transmembrane domain (TMD0) and one ATP hydrolysis site—and recognizes organic anions. However, structural differences between P-gp and MRP1 have not been identified due to the difficulty of resolving the molecular structure of MRP1. To resolve the structure of MRP1, Johnson and Chen performed electron cryomicroscopy (cryo-EM) of bovine MRP1 (bMRP1), which exhibits 91% sequence homology to human MRP1, in the absence or presence of the MRP1 substrate leukotriene C4 (LTC4) to overall resolutions of 3.5 Å and 3.3 Å, respectively. The molecular structure of bMRP1, which was similar to that predicted by sequence and biochemical data, was comprised of the TMD0, an interfacial domain resembling the shape of a lasso, a transporter core similar to that of inward-facing ABC transporters such as P-gp, and two nucleotide binding domains (NBD), NBD1 and NBD2. Although NBD2, which has been previously shown to contain a competent ATPase site, formed a strong interface with its partner TMD2, the NBD1/TMD1 interface was markedly weaker due to the absence of a β-strand s2–α-helix h5 region in NBD1 that is crucial for the docking of a TMD into the cleft on an NBD surface. The cryo-EM–resolved structure of LTC4-bound bMRP1 showed that the binding site of bMRP1 was comprised of a positively charged region and a hydrophobic area, and revealed that substrate binding induced conformational changes in bMRP1 that oriented the NBDs to form a complete ATPase site. These results define the molecular structure of MRP1 and provide insight into the ability of MRP1 to recognize structurally diverse substrates to drive resistance.