Dataset Information

Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.

ABSTRACT: Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na(+) ions, one H(+) and the counter-transport of one K(+) ion. Transport by an archaeal homologue of the human glutamate transporters, Glt(Ph), whose three dimensional structure is known is also coupled to three Na(+) ions but only two Na(+) ion binding sites have been observed in the crystal structure of Glt(Ph). In order to fully utilize the Glt(Ph) structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of Glt(Ph) and accurately determine the number and location of Na(+) ions coupled to transport. Several sites have been proposed for the binding of a third Na(+) ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for Glt(Ph) and reveal a new site for the third Na(+) ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in Glt(Ph), and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na(+) compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na(+) ion in Glt(Ph) and EAAT1.

SUBMITTER: Bastug T

PROVIDER: S-EPMC3302783 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.

Bastug Turgut T Heinzelmann Germano G Kuyucak Serdar S Salim Marietta M Vandenberg Robert J RJ Ryan Renae M RM

PloS one 20120313 3

Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na(+) ions, one H(+) and the counter-transport of one K(+) ion. Transport by an archaeal homologue of the human glutamate transporters, Glt(Ph), whose three dimensional structure is known is also coupled to three Na(+) ions but only two Na(+) ion binding sites have been observed in the crystal structure of Glt(Ph). In order to fully utilize the Glt(Ph) structure in functional studies of t ...[more]

PMID: 22427946

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Project description:The transport cycle in the glutamate transporter (GlT) is catalyzed by the cotransport of three Na(+) ions. However, the positions of only two of these ions (Na1 and Na2 sites) along with the substrate have been captured in the crystal structures reported for both the outward-facing and the inward-facing states of Glt(ph). Characterizing the third ion binding site (Na3) is necessary for structure-function studies attempting to investigate the mechanism of transport in GlTs at an atomic level, particularly for the determination of the sequence of the binding events during the transport cycle. In this study, we report a series of molecular dynamics simulations performed on various bound states of Glt(ph) (the apo state, as well as in the presence of Na(+), the substrate, or both), which have been used to identify a putative Na3 site. The calculated trajectories have been used to determine the water accessibility of potential ion-binding residues in the protein, as a prerequisite for their ion binding. Combined with conformational analysis of the key regions in the protein in different bound states and several additional independent simulations in which a Na(+) ion was randomly introduced to the interior of the transporter, we have been able to characterize a putative Na3 site and propose a plausible binding sequence for the substrate and the three Na(+) ions to the transporter during the extracellular half of the transport cycle. The proposed Na3 site is formed by a set of highly conserved residues, namely, Asp(312), Thr(92), and Asn(310), along with a water molecule. Simulation of a fully bound state, including the substrate and the three Na(+) ions, reveals a stable structure--showing closer agreement to the crystal structure when compared to previous models lacking an ion in the putative Na3 site. The proposed sequence of binding events is in agreement with recent experimental models suggesting that two Na(+) ions bind before the substrate, and one after that. Our results, however, provide additional information about the sites involved in these binding events.

Dataset Information

Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.

Publications

Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.

Similar Datasets

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