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.

Global motions exhibited by proteins in micro- to milliseconds simulations concur with anisotropic network model predictions

“…These observations suggest an inter-subunit coupling that restricts simultaneous opening of subunits. This coupling is reminiscent of the observations made for GltPh simulations in the OF state, 42 which were attributed to intersubunit steric effects restraining the simultaneous opening of all three HP2 gates. The ability of the transport cores of the individual subunits to come into close proximity was confirmed experimentally for the OF state of GltPh.…”

“…Even though various conformations of GltPh have been resolved to date, 15,[34][35][36] and several simulations have been performed using these structural data, 11,[37][38][39][40][41][42][43] the mechanism of function of aspartate transporters (and their mammalian glutamate orthologs) is not yet well-understood. GltPh is a homotrimer.…”

“…Experimental and computational studies have pointed to the role of two hairpins, HP1 and HP2, in gating the substratebinding site. 11,15,35,36,39,42,44 The conformations of HP1 and HP2 will be shown below to be important determinants of the functional substates sampled during two Anton trajectories of 6 μs each, generated for the IF state of GltPh. Strikingly, in line with our findings on BPTI, the essential motions extracted from the Anton simulations for GltPh in the IF state show close overlap with low frequency ANM modes.…”

Evidence for a third sodium-binding site in glutamate transporters suggests an ion/substrate coupling model

“…T370 was not predicted to form part of this Na + -binding site (21). Shrivastava et al (20) noted that T370 (T314 in Glt Ph ) is able to bind water and that this water, in combination with the bound glutamate, could participate in coordinating a Na + ion. Taken together, both thermodynamic and steric considerations point toward the potential location of an ion-binding site in this region, in good agreement with our results from GCMC/MD modeling.…”

“…Earlier theoretical studies also have suggested that the third Na + ion binds in proximity to the bound amino acid substrate (20,21). Holley and Kavanaugh (21), using the knowledge-based method VALE, identified a putative third ion-binding site in which a Na + ion was coordinated by the bound amino acid substrate and three backbone oxygen atoms from HP2.…”