Characterization of the kinetic cycle of an ABC transporter by single-molecule and cryo-EM analyses

• Published in: eLife Vol. 9
• Main Authors: Wang, Ling, Johnson, Zachary Lee, Wasserman, Michael R, Levring, Jesper, Chen, Jue, Liu, Shixin
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 27.05.2020; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: ABC transporter; Active transport; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - metabolism; Animals; Binding Sites; Cancer; Cattle; Characterization; Chemotherapy; conformational dynamics; Cryo-EM; Cryoelectron Microscopy; Drug dosages; Drug resistance; Fluorescence; Fluorescence spectroscopy; Histograms; Hydrolysis; Johnson, Lee; Kinetics; Labeling; Microbial drug resistance; Microscopy; Models, Molecular; MRP1; Multidrug resistance; Multidrug Resistance-Associated Proteins - chemistry; Multidrug Resistance-Associated Proteins - genetics; Multidrug Resistance-Associated Proteins - metabolism; Multidrug resistant organisms; Peptides; Protein Conformation; single-molecule FRET; Spectrometry, Fluorescence; Spectroscopy; Structural Biology and Molecular Biophysics; Time; multidrug resistance; MRP1; conformational dynamics; Cryo-EM; single-molecule FRET; structural biology; none; molecular biophysics; ABC transporter
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.56451

ATP-binding cassette (ABC) transporters are molecular pumps ubiquitous across all kingdoms of life. While their structures have been widely reported, the kinetics governing their transport cycles remain largely unexplored. Multidrug resistance protein 1 (MRP1) is an ABC exporter that extrudes a variety of chemotherapeutic agents and native substrates. Previously, the structures of MRP1 were determined in an inward-facing (IF) or outward-facing (OF) conformation. Here, we used single-molecule fluorescence spectroscopy to track the conformational changes of bovine MRP1 (bMRP1) in real time. We also determined the structure of bMRP1 under active turnover conditions. Our results show that substrate stimulates ATP hydrolysis by accelerating the IF-to-OF transition. The rate-limiting step of the transport cycle is the dissociation of the nucleotide-binding-domain dimer, while ATP hydrolysis per se does not reset MRP1 to the resting state. The combination of structural and kinetic data illustrates how different conformations of MRP1 are temporally linked and how substrate and ATP alter protein dynamics to achieve active transport.