Clinical multidrug resistance in the treatment of bacterial and fungal infections and cancer chemotherapy can result from expression of membrane-embedded efflux pumps that extrude cytotoxic molecules out of the cell. A subclass of these pumps consists of ATP binding cassette (ABC) transporters, ATP-powered traffickers of a wide range of molecules. The long term goal of this research is to define the protein motion that couples energy expenditure to solute translocation by ABC transporters. Roughly 5% of the Escherichia Co//genome encodes for ABC transporters; one of which, MsbA, transduces ATP energy to flip lipid A, the building block of the outer membrane, across the inner membrane. Its critical role in bacterial homeostasis, sequence and functional similarity to human multidrug transporters in conjunction with extensive crystallographic analysis make MsbA a biochemically and biophysically tractable model of clinically relevant ATP-coupled transport. We will use spectroscopic techniques to obtain direct structural and dynamic information on well-defined, key catalytic intermediates of MsbA in lipid bilayers and in the absence of conformational selectivity by crystal lattice forces.
The specific aims willtest a mechanism of transport that envisions anATP- and substrate- regulatedswitch whereby transport occurs concomitantly with cycles of dimerization and dissociation of the nucleotide binding domains (NBDs). Spin labels will be systematically introduced into the protein sequence and their mobilities, accessibilites and pairwise proximities analyzed by electron paramagnetic spectroscopy (EPR) to 1) reconstruct the relative movements of the NBDs and 2) map conformational changes that reorient the substrate binding chamber. A novel aspect of this proposal is the use of complementary spectroscopic rulers with a 5-80A distance range to address controversial aspects of the crystal structures and add a dynamic dimension to these static snapshots. The proposed studies will bridge the current divide between structural and mechanistic models of ABC transporters and provide a unique dynamic perspective on a process of fundamental biochemical importance. In addition to controlling the pharmokinetic profile of xenotoxins, human ABC transporters play causative roles in a number of genetic disorders including cystic fibrosis. Understanding their mechanisms will aid in the design of new drugs to overcome resistance to chemotherapy and the development of therapeutic strategies for the inherited pathologies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biophysics of Synapses, Channels, and Transporters Study Section (BSCT)
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Chin, Jean
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Vanderbilt University Medical Center
Schools of Medicine
United States
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Claxton, Derek P; Jagessar, Kevin L; Steed, P Ryan et al. (2018) Sodium and proton coupling in the conformational cycle of a MATE antiporter from Vibrio cholerae. Proc Natl Acad Sci U S A 115:E6182-E6190
Dastvan, Reza; Fischer, Axel W; Mishra, Smriti et al. (2016) Protonation-dependent conformational dynamics of the multidrug transporter EmrE. Proc Natl Acad Sci U S A 113:1220-5
Martens, Chloé; Stein, Richard A; Masureel, Matthieu et al. (2016) Lipids modulate the conformational dynamics of a secondary multidrug transporter. Nat Struct Mol Biol 23:744-51
Claxton, Derek P; Kazmier, Kelli; Mishra, Smriti et al. (2015) Navigating Membrane Protein Structure, Dynamics, and Energy Landscapes Using Spin Labeling and EPR Spectroscopy. Methods Enzymol 564:349-87
Stein, Richard A; Beth, Albert H; Hustedt, Eric J (2015) A Straightforward Approach to the Analysis of Double Electron-Electron Resonance Data. Methods Enzymol 563:531-67
Masureel, Matthieu; Martens, Chloé; Stein, Richard A et al. (2014) Protonation drives the conformational switch in the multidrug transporter LmrP. Nat Chem Biol 10:149-55
Dürr, Katharina L; Chen, Lei; Stein, Richard A et al. (2014) Structure and dynamics of AMPA receptor GluA2 in resting, pre-open, and desensitized states. Cell 158:778-792
Steed, P Ryan; Stein, Richard A; Mishra, Smriti et al. (2013) Na?-substrate coupling in the multidrug antiporter norm probed with a spin-labeled substrate. Biochemistry 52:5790-9
Alexander, Nathan S; Stein, Richard A; Koteiche, Hanane A et al. (2013) RosettaEPR: rotamer library for spin label structure and dynamics. PLoS One 8:e72851
Steed, P Ryan; Zou, Ping; Trone, Kristin E et al. (2013) Structure and pH-induced structural rearrangements of the putative multidrug efflux pump EmrD in liposomes probed by site-directed spin labeling. Biochemistry 52:7964-74

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