TY - JOUR
T1 - Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis
AU - Riederer, Erika A.
AU - Valiyaveetil, Francis I.
N1 - Funding Information:
This research was supported by NIH Grants R01GM087546 and R37NS085318 (to Dr. Olga Boudker, Principal Investigator and F.I.V., Coinvestigator). E.A.R. was supported by a Predoctoral Fellowship from the American Heart Association (AHA 19PRE34380950).
Funding Information:
While the overall binding scheme is supported by this study and previous investigations, the mechanisms underlying key steps are presently unresolved. These include the molecular details of how the R397 side chain in the Apo conformation keeps HP2 closed or how the M311 conformational switch allows HP2 to open. High-resolution structure determination of the various states combined with focused computational studies will be necessary to understand these mechanisms.
Publisher Copyright:
© 2019 National Academy of Sciences. All rights reserved.
PY - 2019/8/6
Y1 - 2019/8/6
N2 - Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, GltPh is an archaeal homolog of glutamate transporters and has been extensively used to understand the transport mechanism. A critical aspect of the transport cycle in GltPh is the coupled binding of sodium and aspartate. Previous studies have suggested a major role for hairpin-2 (HP2), which functions as the extracellular gate for the aspartate binding site, in the coupled binding of sodium and aspartate to GltPh. In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh. We use the HP2 assays to show that HP2 opening with Na+ follows an induced-fit mechanism. We also determine how residues in the substrate binding site affect the opening and closing of HP2.
AB - Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, GltPh is an archaeal homolog of glutamate transporters and has been extensively used to understand the transport mechanism. A critical aspect of the transport cycle in GltPh is the coupled binding of sodium and aspartate. Previous studies have suggested a major role for hairpin-2 (HP2), which functions as the extracellular gate for the aspartate binding site, in the coupled binding of sodium and aspartate to GltPh. In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh. We use the HP2 assays to show that HP2 opening with Na+ follows an induced-fit mechanism. We also determine how residues in the substrate binding site affect the opening and closing of HP2.
KW - Fluorescence
KW - Glutamate transporters
KW - Unnatural amino acids
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U2 - 10.1073/pnas.1907852116
DO - 10.1073/pnas.1907852116
M3 - Article
C2 - 31332002
AN - SCOPUS:85070200676
SN - 0027-8424
VL - 116
SP - 15939
EP - 15946
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
IS - 32
ER -