Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 116, Issue 32, Pages 15939-15946Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1907852116
Keywords
glutamate transporters; unnatural amino acids; fluorescence
Categories
Funding
- NIH [R01GM087546, R37NS085318]
- American Heart Association [AHA 19PRE34380950]
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Glutamate transporters harness the ionic gradients across cell membranes for the concentrative uptake of glutamate. The sodium-coupled Asp symporter, Glt(ph )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 Glt(ph) 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 Glt(ph). In this study, we develop a fluorescence assay for monitoring HP2 movement by incorporating tryptophan and the unnatural amino acid, p-cyanophenylalanine into Glt(ph). 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. Our data, combined with previous studies, provide the molecular sequence of events in the coupled binding of sodium and aspartate to Glt(ph).
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