TY - JOUR
T1 - Electrostatic lock in the transport cycle of the multidrug resistance transporter EmrE
AU - Vermaas, Josh V.
AU - Rempe, Susan B.
AU - Tajkhorshid, Emad
N1 - Funding Information:
ACKNOWLEDGMENTS. We acknowledge Katherine Henzler-Wildman, Nate Traaseth, Chao Wu, and Reza Dastvan for helpful conversations that guided the research direction. Additionally, we thank Reza Dastvan and Hassane Mchaourab for providing the spin-label distances in a text format for comparison with our own results. J.V.V. and S.B.R. gratefully acknowledge support from the Sandia National Laboratories (SNL) Campus Executive Program, which is funded by the Laboratory Directed Research and Development Program. This project made extensive use of the high-performance computing resources provided by SNL. The project also received support
Funding Information:
We acknowledge Katherine Henzler-Wildman, Nate Traaseth, Chao Wu, and Reza Dastvan for helpful conversations that guided the research direction. Additionally, we thank Reza Dastvan and Hassane Mchaourab for providing the spin-label distances in a text format for comparison with our own results. J.V.V. and S.B.R. gratefully acknowledge support from the Sandia National Laboratories (SNL) Campus Executive Program, which is funded by the Laboratory Directed Research and Development Program. This project made extensive use of the high-performance computing resources provided by SNL. The project also received support from the Defense Threat Reduction Agency–Joint Science and Technology Office for Chemical and Biological Defense (Interagency Agreement DTRA10027IA-3167, to S.B.R.). SNL is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia LLC, a wholly owned subsidiary of Honeywell International Inc. for the US Department of Energy’s (DOE) National Nuclear Security Administration under Contract DE-NA0003525. This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the US DOE’s Office of Science by Los Alamos National Laboratory (Contract DE-AC52-06NA25296) and SNL. This research is also supported by the National Institutes of Health, through Grants P41-GM104601 and U54-GM087519 (to E.T.). The views expressed in this article do not necessarily represent the views of the US DOE or the US government.
Funding Information:
from the Defense Threat Reduction Agency–Joint Science and Technology Office for Chemical and Biological Defense (Interagency Agreement DTRA10027IA-3167, to S.B.R.). SNL is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia LLC, a wholly owned subsidiary of Honeywell International Inc. for the US Department of Energy’s (DOE) National Nuclear Security Administration under Contract DE-NA0003525. This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the US DOE’s Office of Science by Los Alamos National Laboratory (Contract DE-AC52-06NA25296) and SNL. This research is also supported by the National Institutes of Health, through Grants P41-GM104601 and U54-GM087519 (to E.T.). The views expressed in this article do not necessarily represent the views of the US DOE or the US government.
Publisher Copyright:
© 2018 National Academy of Sciences. All rights reserved.
PY - 2018/8/7
Y1 - 2018/8/7
N2 - EmrE is a small, homodimeric membrane transporter that exploits the established electrochemical proton gradient across the Escherichia coli inner membrane to export toxic polyaromatic cations, prototypical of the wider small-multidrug resistance transporter family. While prior studies have established many fundamental aspects of the specificity and rate of substrate transport in EmrE, low resolution of available structures has hampered identification of the transport coupling mechanism. Here we present a complete, refined atomic structure of EmrE optimized against available cryo-electron microscopy (cryo-EM) data to delineate the critical interactions by which EmrE regulates its conformation during the transport process. With the model, we conduct molecular dynamics simulations of the transporter in explicit membranes to probe EmrE dynamics under different substrate loading and conformational states, representing different intermediates in the transport cycle. The refined model is stable under extended simulation. The water dynamics in simulation indicate that the hydrogen-bonding networks around a pair of solvent-exposed glutamate residues (E14) depend on the loading state of EmrE. One specific hydrogen bond from a tyrosine (Y60) on one monomer to a glutamate (E14) on the opposite monomer is especially critical, as it locks the protein conformation when the glutamate is deprotonated. The hydrogen bond provided by Y60 lowers the pKa of one glutamate relative to the other, suggesting both glutamates should be protonated for the hydrogen bond to break and a substrate-free transition to take place. These findings establish the molecular mechanism for the coupling between proton transfer reactions and protein conformation in this proton-coupled secondary transporter.
AB - EmrE is a small, homodimeric membrane transporter that exploits the established electrochemical proton gradient across the Escherichia coli inner membrane to export toxic polyaromatic cations, prototypical of the wider small-multidrug resistance transporter family. While prior studies have established many fundamental aspects of the specificity and rate of substrate transport in EmrE, low resolution of available structures has hampered identification of the transport coupling mechanism. Here we present a complete, refined atomic structure of EmrE optimized against available cryo-electron microscopy (cryo-EM) data to delineate the critical interactions by which EmrE regulates its conformation during the transport process. With the model, we conduct molecular dynamics simulations of the transporter in explicit membranes to probe EmrE dynamics under different substrate loading and conformational states, representing different intermediates in the transport cycle. The refined model is stable under extended simulation. The water dynamics in simulation indicate that the hydrogen-bonding networks around a pair of solvent-exposed glutamate residues (E14) depend on the loading state of EmrE. One specific hydrogen bond from a tyrosine (Y60) on one monomer to a glutamate (E14) on the opposite monomer is especially critical, as it locks the protein conformation when the glutamate is deprotonated. The hydrogen bond provided by Y60 lowers the pKa of one glutamate relative to the other, suggesting both glutamates should be protonated for the hydrogen bond to break and a substrate-free transition to take place. These findings establish the molecular mechanism for the coupling between proton transfer reactions and protein conformation in this proton-coupled secondary transporter.
KW - Membrane protein
KW - Molecular dynamics
KW - Proton-coupled transport
KW - Structure refinement
UR - http://www.scopus.com/inward/record.url?scp=85051187413&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85051187413&partnerID=8YFLogxK
U2 - 10.1073/pnas.1722399115
DO - 10.1073/pnas.1722399115
M3 - Article
C2 - 30026196
AN - SCOPUS:85051187413
VL - 115
SP - E7502-E7511
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 32
ER -