Gating motions in the outward- and inward-facing conformations of EAAT3.

<p>Time evolution of the S332(C)–V411(C) distance in the outward-facing conformation with (A) no ligands bound to the transporter, and (B) only the Na1 and Na3 ions bound to the transporter. The same for the inward-facing conformation with (C) no ligands bound to the transporter, and (D) only the Na1 and Na3 ions bound to the transporter. The color code for the subunits are A (black), B (red), and C (green).</p

Histogram analysis of the distance between the C<i>α</i> atoms of T70 and D49 obtained from the MD simulations of HL[D67H] at 300 K and 400 K.

<p>The last 50 ns of the trajectory data are used in the analysis.</p

Bottom view of the complex structure demonstrating blocking of the pore.

<p>The toxin residues R13, K11, and K16 make contacts with the channel residues EEDD in all four domains.</p

Binding free energies for K⁺ and K⁺/Na⁺ selectivity free energies at the three sites proposed for the inward-facing EAAT3.

<p>The forward and the backward calculations of the interaction energy are listed in the second and third columns, respectively, and their average in the fourth column. The total binding free energy that includes the translational free energy difference (fifth column) is given in the last column. Errors are estimated from block data analysis using 100 ps windows. All energies are in kcal/mol.</p

Snapshots of the stable binding modes obtained from the MD simulations for (A) Complex-1, (B) Complex-2, and (C) Complex-4.

<p>The side chains involved in the binding are shown explicitly (labeled with double prime in monomer-2 on the right).</p

Convergence study of the Na_v1.4–<i>µ</i>-GIIIA PMF from block data analysis.

<p>To minimize fluctuations, a relatively large sampling size of 2 ns is used, which is slided in 1 ns steps over the 10 ns of data. The block-data PMFs drop monotonically in the first 4 ns as the system equilibrates and then fluctuate around a base line from 4–10 ns, signalling equilibration. The final PMF obtained from 4–10 ns is indicated by a thick black line.</p

Molecular Dynamics Simulations of the Mammalian Glutamate Transporter EAAT3

<div><p>Excitatory amino acid transporters (EAATs) are membrane proteins that enable sodium-coupled uptake of glutamate and other amino acids into neurons. Crystal structures of the archaeal homolog Glt_Ph have been recently determined both in the inward- and outward-facing conformations. Here we construct homology models for the mammalian glutamate transporter EAAT3 in both conformations and perform molecular dynamics simulations to investigate its similarities and differences from Glt_Ph. In particular, we study the coordination of the different ligands, the gating mechanism and the location of the proton and potassium binding sites in EAAT3. We show that the protonation of the E374 residue is essential for binding of glutamate to EAAT3, otherwise glutamate becomes unstable in the binding site. The gating mechanism in the inward-facing state of EAAT3 is found to be different from that of Glt_Ph, which is traced to the relocation of an arginine residue from the HP1 segment in Glt_Ph to the TM8 segment in EAAT3. Finally, we perform free energy calculations to locate the potassium binding site in EAAT3, and find a high-affinity site that overlaps with the Na1 and Na3 sites in Glt_Ph.</p></div

Number of water molecules in the hydrophobic pocket of HL and HL[D67H].

<p>Number of water molecules in the hydrophobic pocket of HL and HL[D67H].</p

Effect of different mutations on the properties of EAAT1, EAAT2 (GLT-1) and EAAT3 (EAAC1) transporters.

<p>Transporter/Exchanger functionality is given in the third and fourth columns. Columns 5 and 6 gives the Na⁺ affinity in the presence and absence of glutamate, respectively. Columns 7 and 8 specify K⁺ and Li⁺ interactions with the transporter. Glu and Asp affinities are given in the last two columns. For the affinities, N/S means no saturation of the binding site in the range considered, and nRed/nInc means reduction/increase in the ligand affinity by orders of magnitude (0, same order). Human sequences are used for EAAT3 which have one extra residue than the rat sequences employed in some experiments.</p><p>*All glutamate affinities measured at [Na⁺] between 100–150 mM.</p>†<p>Result for D-aspartate.</p>‡<p>Enables succinate exchange, unlike wild-type.</p

QMEAN scores and DFIRE energy values for the Glt_Ph crystal structures, and the EAAT3 models constructed in this work.

<p>The QMEAN score goes from 0 to 1, 1 being the best possible model. Lower DFIRE energy values indicate better quality structures.</p