Substrate Diffusion and Binding to Glutamate Transporter
Achieved Within Nanoseconds
Indira Shrivastava, Susan Amara & Ivet Bahar
(J Biol Chem article (html) (pdf) ; Supplementary Material)
An important advancement towards a molecular understanding
of the mechanism of function of glutamate transporters has been the recent determination
of the X-ray structure of the archeal glutamate transporter (Gltph)
from Pyrococcus horikoshii. This structure
supports a model in which the substrate binding site is located between two hairpin loops
HP1 and HP2 proposed to serve as internal and external gates of the transport
pathway (Yernool et al, 2004), (Boudker et al, 2007).
Although the original X-ray structure (Yernool et al, 2004)was considered to
be in the glutamate-bound form (4), its resolution could not permit to
visualize the substrate at the binding site. This structure served, however, as
an excellent starting point for further exploring the molecular dynamics of glutamate
Molecular Dynamics simulations of this
protein, in a solvated membrane bilayer, revealed 'flipper-like' movements,
exposing polar residues at the binding site, which serve as an attractor for driving
the diffusion of glutamate within nanoseconds.MD_Movies
At the binding site, glutamate remains
locked at that position by a network of hydrogen bonds involving a highly
conserved serine motif (Ser277-Ser279) on HP1 and two glycines (Gly354 and
Gly357) on HP2, consistent with biochemical experiments.
Diffusion of glutamate towards the subunit with the open EC gate and resulting bound structure. Panel A displays the minimum distance between glutamate and selected residues/motifs at the binding site, observed in MD1. Binding site is initiated by the interaction with conserved glycines, G354-G357 (red) on HP2 followed by T275-S279 on HP1 tip (black). Approximately 0.5ns later, glutamate approaches the TM7 residue D312-T314 (blue) and TM8 residue D394 (orange), R397-T398 (green) and D390 (magenta). The snapshots of the protein and substrate (encircled) at 0ps (top) and 4ns (bottom) are shown. One subunit is omitted for clarity. The curves provide information on the series of events that gradually direct and stabilize the substrate. HP1 and HP2 tip are instrumental in driving the diffusion. The inset displays the residues on HP1 and HP2 residues which interact closely with Glutamate. Panel B displays the selected residues/motifs whose distances from Glutamate are displayed in panel A.
Substrate binding triggers an orderly entry of water molecules into the binding site mediated by
two highly conserved aspartic acids (D390 and D394) on TM8.
Six snapshots from MD1 are shown, starting from t = 0 (upper, left) and ending at 4 ns (bottom, right). The trajectory of five water molecules originally dispersed at various locations in the EC environment is shown. The first water molecules enters through the opening between HP2 and HP1 to bind T314 on TM7 (see Supplementary Material, Fig 3 and movies 2 and 3), and remains at that position after glutamate binds (around 1 ns) and locks the entry/exit pathway. The remaining four are positioned to orderly enter the binding site through another interstice after 2.5 ns (snapshot at 3.55 ns), attracted by D390 and D394 on TM8. Successive entry of all water molecules takes place in an orderly fashion through the same pathway within 0.3 ns, resulting in the tight interaction between substrate and water molecules in the binding site, on which the HP2 closes down. The glutamate remains tightly bound in the subsequent 6 ns simulations, while the binding site is solvated at all times by a continuous circulation of water molecules.
molecules further stabilize the glutamate at the binding site. Two
Na+ binding sites are also identified in the core, one close to the
substrate binding site, and the second deeper down towards the intracellular
which agree remarkably well with the sites recently reported by Gouaux
and coworkers (Boudker et al. (2007) (pdf)
An excellent agreement with MD simulations and Xray structure are
observed, w.r.t ligand-protein interactions.
Residues implicated in transport and/or binding in Gltph