Dynamics of the extracellular gate and ion-substrate coupling in the glutamate transporter - PubMed (original) (raw)

Dynamics of the extracellular gate and ion-substrate coupling in the glutamate transporter

Zhijian Huang et al. Biophys J. 2008 Sep.

Abstract

Glutamate transporters (GluTs) are the primary regulators of extracellular concentration of the neurotransmitter glutamate in the central nervous system. In this study, we have investigated the dynamics and coupling of the substrate and Na(+) binding sites, and the mechanism of cotransport of Na(+) ions, using molecular dynamics simulations of a membrane-embedded model of GluT in its apo (empty form) and various Na(+)- and/or substrate-bound states. The results shed light on the mechanism of the extracellular gate and on the sequence of binding of the substrate and Na(+) ions to GluT during the transport cycle. The results suggest that the helical hairpin HP2 plays the key role of the extracellular gate for the substrate binding site, and that the opening and closure of the gate is controlled by substrate binding. GluT adopts an open conformation in the absence of the substrate exposing the binding sites of the substrate and Na(+) ions to the extracellular solution. Based on the calculated trajectories, we propose that Na1 is the first element to bind GluT, as it is found to be important for the completion of the substrate binding site. The subsequent binding of the substrate, in turn, is shown to result in an almost complete closure of the extracellular gate and the formation of the Na2 binding site. Finally, binding of Na2 locks the extracellular gate and completes the formation of the occluded state of GluT.

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Figures

FIGURE 1

Relevant structural features of GluT. (a) The simulation system used in this study is composed of the bowl-shaped trimer of GluT embedded in a lipid bilayer and water. (b) The structure of GluT monomer with bound substrate (shown in VDW) and the two structurally resolved Na+ ions (spheres). Helical hairpins HP1 and HP2, and transmembrane helices TM7 and TM8, which, together, form the substrate binding site, are shown in a darker shade.

FIGURE 2

Dynamics of the extracellular gate in GluT. Left and right panels show the results of the simulations performed in the presence and in the absence of the substrate, respectively. See Table 1 for the description of the systems. (a) The last frame of simulation S1A showing the occluded state of GluT. (b) Opening of the substrate binding site upon removal of the substrate is mediated through a large displacement of HP2 and its separation from HP1, as captured, e.g., in one of the last frames of simulation S2B. (c_–_f) Time evolution of the RMSDs of the helical hairpins HP1 and HP2. Panels c and d show the RMSDs of HP1, and panels e and f show the RMSDs of HP2. (g and h) Time evolution of the distance between HP1 and HP2, measured as the distance between S277(C_α_) and G355(C_α_), located respectively at the tips of HP1 and HP2. A clear dependence of the dynamics of HP2 on the presence of the substrate is evident.

FIGURE 3

Coupling of binding of the two Na+ ions and the substrate. (a and b) Substrate-induced formation of Na2 binding site. Formation of the Na2 binding site upon substrate binding is shown by comparing the structure of the binding site in the absence (a, simulation S2B) and in the presence (b, simulation S2A) of the substrate. Blue arrows represent the dipole moments of the half-helices TM7a and HP2a. The dipole moments are misaligned in the absence of the substrate, but exhibit a significant alignment upon substrate binding, resulting in the formation of the Na2 binding site (marked with a circle in b). (c) Comparison of the binding site of the fully open state from the simulations (last snapshot of the simulation of the apo form, S2B, shown in the same colors as in other panels) with the crystal structure of the occluded state (in the presence of the substrate and the two Na+ ions, shown in white) and the crystal structure of the open state (bound with TBOA, shown in violet). (d_–_f) Na2-induced formation of the occluded state. Binding of Na2 to GluT results in complete closure of the binding site to the extracellular solution. In the absence of Na2 (d, simulation S2A), the binding pocket is accessible to water, whereas, upon Na2 binding (e, simulation S1C), the binding site is completely sealed. An overlay of the two states (colored, before Na2 binding; white, after Na2 binding) is shown in panel f highlighting the small change in the conformation of HP2 upon Na2 binding. (g_–_i) Indirect effect of Na1 on the substrate binding site. (g) The substrate binding site in the occluded state of GluT showing all residues that directly interact with the substrate. (h) In the absence of Na1, the side chain of N401 leaves the substrate binding site due to direct interactions with N310. (i) Na1 binding to GluT results in the upward shift of N401 and the recovery of the substrate binding site.

FIGURE 4

Hypothetical mechanism for binding of the substrate and the two structurally resolved Na+ ions during the extracellular half of the transport cycle in GluT. Substrate is shown in licorice. Na+ ions are represented by spheres. (a) Na1 binding to GluT results in the complete formation of the substrate binding site. (b) The substrate binds to GluT and partially closes the extracellular gate HP2, resulting in the formation of the Na2 binding site. (c) Finally, Na2 enters its newly formed binding site from the extracellular side, and locks the extracellular gate in its fully closed form, resulting in the formation of the occluded state. The molecular images are made using the last snapshots of the simulations S2B (apo GluT), S3B (GLuT + Na1), S1B (GluT + Na1 + substrate), and S1A (GluT + Na1 + Na2 + substrate), respectively.

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Dynamics of the extracellular gate and ion-substrate coupling in the glutamate transporter - PubMed (original) (raw)