Molecular simulations elucidate the substrate translocation pathway in a glutamate transporter

Abstract

Glutamate transporters are membrane proteins found in neurons and glial cells, which play a critical role in regulating cell signaling by clearing glutamate released from synapses. Although extensive biochemical and structural studies have shed light onto different aspects of glutamate transport, the time-resolved molecular mechanism of substrate (glutamate or aspartate) translocation, that is, the sequence of events occurring at the atomic level after substrate binding and before its release intracellularly remain to be elucidated. We identify an energetically preferred permeation pathway of Ϸ23 Å between the helix HP1b on the hairpin HP1 and the transmembrane helices TM7 and TM8, using the high resolution structure of the transporter from Pyrococcus horikoshii (Glt Ph ) in steered molecular dynamics simulations. Detailed potential of mean force calculations along the putative pathway reveal 2 energy barriers encountered by the substrate (aspartate) before it reaches the exit. The first barrier is surmounted with the assistance of 2 conserved residues (S278 and N401) and a sodium ion (Na2); and the second, by the electrostatic interactions with D405 and another sodium ion (Na1). The observed critical interactions and mediating role of conserved residues in the core domain, the accompanying conformational changes (in both substrate and transporter) that relieve local strains, and the unique coupling of aspartate transport to Na ؉ dislocation provide insights into methods for modulating substrate transport.

Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats

“…4A) (13). To date, proposed mechanisms of the conformational change required to expose this aspartate binding site to the cytoplasmic solution have invoked relatively small movements in the protein, involving mainly HP1, with the rest of the structure remaining essentially unchanged (6,7,19). Our atomistic model suggests that the cytoplasm-facing conformation of GltPh is a symmetry-related version of the extracellularfacing structures, and that the inward-and outward-facing states interchange through a large, concerted movement of the core binding region of HP1, TM7, HP2, and TM8 (see Figs.…”

Mechanism of Substrate Translocation in an Alternating Access Transporter

“…This reflects not only the relative scarcity of experimental structures of transporters but also the large size of many transporters and the intensive computational requirements of MD simulations on the relatively long timescales at which transport occurs. Several elegant studies have provided important insights into these events through atomic-level simulations by relying on biased or guided simulation techniques (Cheng and Bahar, 2014; Gu et al, 2009; Liu et al, 2015; Moradi et al, 2015; Moradi and Tajkhorshid, 2013; Shimamura et al, 2010), but the artificial forces generally used to accelerate transitions in such simulations might give rise to unphysical transition pathways, complicating the mechanistic interpretation of these simulations.…”

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

“…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.…”