Abstract
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.
Highlights
Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette (ABC) transporter that harnesses the energy of ATP to extrude substrates from the cytosol to the extracellular space (Cole, 2014a)
Using cryo-EM and mutagenesis, we captured three snapshots of MRP1, demonstrating the conformational changes induced by leukotriene C4 (LTC4) and ATP (Johnson and Chen, 2017; Johnson and Chen, 2018)
Here we employed single-molecule fluorescence spectroscopy to characterize the dynamics of MRP1 to understand the connectivity of these conformations
Summary
Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette (ABC) transporter that harnesses the energy of ATP to extrude substrates from the cytosol to the extracellular space (Cole, 2014a). Structures of bovine MRP1 (bMRP1) have been determined by electron cryo-microscopy (cryo-EM) in three functional states (Johnson and Chen, 2017; Johnson and Chen, 2018): an apo form in the absence of substrate and ATP, a complex with the native substrate leukotriene C4 (LTC4) in the absence of ATP, and a structure of the hydrolysis-deficient E1454Q mutant determined in the presence of both LTC4 and ATP (Figure 1A) These structures, in accord with decades of functional analysis (Cole, 2014a) bring about the following understanding of the transport cycle. The LTC4-binding pocket becomes deformed, no longer competent to bind substrate In this outward-facing (OF) conformation, two ATP molecules are occluded at the NBD dimer interface: one in the catalytically inactive, degenerate site and the other in the active consensus site, poised for hydrolysis (Figure 1A). The synergy between the smFRET and cryo-EM studies has enabled us to determine the rate-limiting mechanism of MRP1 and how substrate and ATP modulate the kinetics of this important drug transporter
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