Research will continue on the long term goal of defining the conformational motion that couples energy expenditure to substrate translocation in active multidrug transporters. Clinical multidrug resistance in the treatment of bacterial and fungal infections and chemotherapy of neoplasms can be associated with overexpression of these membrane-embedded efflux pumps that selectively extrude cytotoxic molecules from the cell. The experimental focus for the next funding period is on two superfamilies, the ATP binding cassettes (ABC) and the major facilitator (MFS), that account for the majority of bacterial multidrug resistance transporters, represent two energy conversion motifs and encompass a broad spectrum of extruded drugs. ABC transporters harness the energy of ATP hydrolysis to power transport while MFS transporters couple substrate translocation to inward movement of protons. An innovative experimental design combines quantitative ensemble analysis by advanced spin labeling electron paramagnetic resonance (EPR) methods with insight into long range motions by disulfide chemistry, to derive constraints that describe the conformational state of each transporter at different stages of the transport cycle. We will test whether a model of ATP-induced conformational changes, developed in the previous funding period, describes the transport cycle of ABC drug efflux transporters in a native-like environment. For multidrug transporters of the major facilitator superfamily, we will investigate the structural basis of proton/substrate coupling and delineate the common structural motifs underlying transporter isomerization from inward-facing to outward-facing conformations. The experiments will capitalize on the convergence of two technologies, Nanodiscs phospholipid bilayers and Q-band pulse EPR, to increase the throughput of long range distance measurements (up to 70E) between spin labels by double electron electron resonance (DEER). The successful implementation of the proposed methodology will set the stage for application to eukaryotic membrane proteins where absolute amounts and concentrations are more limited. The results will provide the dynamic dimension necessary to bridge the divide between functional models of these transporters and static crystallographic snapshots that are often mechanistically ill-defined.
The World Health Organization has reported that multidrug-resistant bacteria account for up to 60% of all hospital-acquired infections globally. The major outcome of this research is to learn common principles of how multidrug transporters harness energy input for vectorial substrate movement. These are fundamental information for the development of new therapeutic strategies to combat the evolving epidemic of drug resistance and overcome tumor resistance to chemotherapy.
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|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|
|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|
|Masureel, Matthieu; Martens, ChloÃ©; Stein, Richard A et al. (2014) Protonation drives the conformational switch in the multidrug transporter LmrP. Nat Chem Biol 10:149-55|
|DÃ¼rr, 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 158:778-92|
|Steed, P Ryan; Stein, Richard A; Mishra, Smriti et al. (2013) Naâº-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|
|Hirst, Stephanie J; Alexander, Nathan; McHaourab, Hassane S et al. (2011) RosettaEPR: an integrated tool for protein structure determination from sparse EPR data. J Struct Biol 173:506-14|
|Kazmier, Kelli; Alexander, Nathan S; Meiler, Jens et al. (2011) Algorithm for selection of optimized EPR distance restraints for de novo protein structure determination. J Struct Biol 173:549-57|
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