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Heterogeneous substrate binding in a glutamate transporter homologue

View ORCID ProfileKrishna D. Reddy, View ORCID ProfileDidar Ciftci, Amanda Scopelliti, View ORCID ProfileOlga Boudker
doi: https://doi.org/10.1101/2021.07.08.451670
Krishna D. Reddy
1Department of Physiology and Biophysics, Weill Cornell Medicine, 1300 York Avenue, 10065, NY, USA
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  • ORCID record for Krishna D. Reddy
Didar Ciftci
1Department of Physiology and Biophysics, Weill Cornell Medicine, 1300 York Avenue, 10065, NY, USA
2Tri-Institutional Training Program in Chemical Biology, New York, NY 10064, USA
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Amanda Scopelliti
1Department of Physiology and Biophysics, Weill Cornell Medicine, 1300 York Avenue, 10065, NY, USA
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Olga Boudker
1Department of Physiology and Biophysics, Weill Cornell Medicine, 1300 York Avenue, 10065, NY, USA
3Howard Hughes Medical Institute
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  • For correspondence: olb2003@med.cornell.edu
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ABSTRACT

Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this family, including kinetic models of transport. Recent studies have revealed transport rate heterogeneity in an archaeal glutamate transporter homologue GltPh, inconsistent with simple kinetic models. The structural and mechanistic determinants of this heterogeneity remain undefined. In a mutant form of GltPh, we demonstrate substrate binding heterogeneity in the outward-facing state, modulated by temperature and salts. We observe similar trends in wild-type GltPh that correlate with changes in the transport rate. Extensive cryo-EM analysis of the fully bound mutant GltPh provides multiple potential explanations of heterogeneous substrate binding. At equilibrium, we show subtle differences in tilts of protomers in the outward-facing state and configurations of the substrate-binding pocket. Within seconds of substrate binding, we observe perturbed helical packing of the extracellular half of the substrate-binding domain. Some or all of these may contribute to the heterogeneity observed in binding and transport.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted July 09, 2021.
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Heterogeneous substrate binding in a glutamate transporter homologue
Krishna D. Reddy, Didar Ciftci, Amanda Scopelliti, Olga Boudker
bioRxiv 2021.07.08.451670; doi: https://doi.org/10.1101/2021.07.08.451670
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Heterogeneous substrate binding in a glutamate transporter homologue
Krishna D. Reddy, Didar Ciftci, Amanda Scopelliti, Olga Boudker
bioRxiv 2021.07.08.451670; doi: https://doi.org/10.1101/2021.07.08.451670

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