title: Time-resolved mechanism of extracellular gate opening and substrate binding in a glutamate transporter.
abstract: Glutamate transporters, also referred to as excitatory amino acid transporters (EAATs), are membrane proteins that regulate glutamatergic signal transmission by clearing excess glutamate after its release at synapses. A structure-based understanding of their molecular mechanisms of function has been elusive until the recent determination of the x-ray structure of an archaeal transporter, Glt(Ph). Glt(Ph) exists as a trimer, with each subunit containing a core region that mediates substrate translocation. In the present study a series of molecular dynamics simulations have been conducted and analyzed in light of new experimental data on substrate binding properties of EAATs. The simulations provide for the first time a full atomic description of the time-resolved events that drive the recognition and binding of substrate. The core region of each subunit exhibits an intrinsic tendency to open the helical hairpin HP2 loop, the extracellular gate, within tens of nanoseconds exposing conserved polar residues that serve as attractors for substrate binding. The NMDGT motif on the partially unwound part of the transmembrane helix TM7 and the residues Asp-390 and Asp-394 on TM8 are also distinguished by their important role in substrate binding and close interaction with mediating water molecules and/or sodium ions. The simulations reveal a Na+ binding site comprised in part of Leu-303 on TM7 and Asp-405 on TM8 and support a role for sodium ions in stabilizing substrate-bound conformers. The functional importance of Leu-303 or its counterpart Leu-391 in human EAAT1 (hEAAT1) is confirmed by site-directed mutagenesis and Na+ dependence assays conducted with hEAAT1 mutants L391C and L391A.
authors.affiliation: Department of Computational Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
authors.forename: Indira H
authors.initials: IH
authors.lastname: Shrivastava
authors.forename: Jie
authors.initials: J
authors.lastname: Jiang
authors.forename: Susan G
authors.initials: SG
authors.lastname: Amara
authors.forename: Ivet
authors.initials: I
authors.lastname: Bahar
mesh_terms: D020816:Amino Acid Motifs; D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D001665:Binding Sites; D003198:Computer Simulation; D018360:Crystallography, X-Ray / Q000379:methods; D005971:Glutamates / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D012964:Sodium / Q000737:chemistry; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D027322:Amino Acid Transport System X-AG; D005971:Glutamates; D008051:Lipid Bilayers; D018698:Glutamic Acid; D012964:Sodium