Investigation of the allosteric coupling mechanism in a glutamate transporter homolog via unnatural amino acid mutagenesis.

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  • Author(s): Riederer EA;Riederer EA; Valiyaveetil FI; Valiyaveetil FI
  • Source:
    Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2019 Aug 06; Vol. 116 (32), pp. 15939-15946. Date of Electronic Publication: 2019 Jul 22.
  • Publication Type:
    Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: National Academy of Sciences Country of Publication: United States NLM ID: 7505876 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1091-6490 (Electronic) Linking ISSN: 00278424 NLM ISO Abbreviation: Proc Natl Acad Sci U S A Subsets: MEDLINE
    • Publication Information:
      Original Publication: Washington, DC : National Academy of Sciences
    • Subject Terms:
      Sequence Homology, Amino Acid*; Amino Acid Transport System X-AG/*genetics ; Amino Acid Transport System X-AG/*metabolism ; Amino Acids/*genetics ; Mutagenesis/*genetics; Allosteric Regulation ; Amino Acid Transport System X-AG/chemistry ; Fluorescence ; Kinetics ; Models, Molecular ; Protein Structure, Secondary ; Sodium/metabolism
    • Abstract:
      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 .
      Competing Interests: The authors declare no conflict of interest.
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    • Grant Information:
      R01 GM087546 United States GM NIGMS NIH HHS; R21 NS113561 United States NS NINDS NIH HHS; R37 NS085318 United States NS NINDS NIH HHS
    • Contributed Indexing:
      Keywords: fluorescence; glutamate transporters; unnatural amino acids
    • Accession Number:
      0 (Amino Acid Transport System X-AG)
      0 (Amino Acids)
      9NEZ333N27 (Sodium)
    • Publication Date:
      Date Created: 20190724 Date Completed: 20200330 Latest Revision: 20200915
    • Publication Date:
      20221213
    • Accession Number:
      PMC6690018
    • Accession Number:
      10.1073/pnas.1907852116
    • Accession Number:
      31332002