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To whom correspondence should be addressed: Dept. of Computational and Systems Biology, School of Medicine, University of Pittsburgh, 3064 BST3, 3501 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-648-3332; Fax: 412-648-3163;
* This work was supported, in whole or in part, by National Institutes of Health Grants R01GM086238 from the NIGMS (to I. B.) and R01 LM007994-08 (to I. B.) and the National Institutes of Health-funded Biomedical Technology Research Center (Grant P41 GM103712) (to I. B.). This article contains supplemental Table S1, Figs. S1–S5, and supplemental Movie S1, A and B.
Sodium-coupled neurotransmitter transporters play a key role in neuronal signaling by clearing excess transmitter from the synapse. Structural data on a trimeric archaeal aspartate transporter, GltPh, have provided valuable insights into structural features of human excitatory amino acid transporters. However, the time-resolved mechanisms of substrate binding and release, as well as that of coupling to sodium co-transport, remain largely unknown for this important family. We present here the results of the most extensive simulations performed to date for GltPh in both outward-facing and inward-facing states by taking advantage of significant advances made in recent years in molecular simulation technology. The generated multiple microsecond trajectories consistently show that the helical hairpin HP2, not HP1, serves as an intracellular gate (in addition to its extracellular gating role). In contrast to previous proposals, HP1 can neither initiate nor accommodate neurotransmitter release without prior opening of HP2 by at least 4.0 Å. Aspartate release invariably follows that of a sodium ion located near the HP2 gate entrance. Asp-394 on TM8 and Arg-276 on HP1 emerge as key residues that promote the reorientation and diffusion of substrate toward the cell interior. These findings underscore the significance of examining structural dynamics, as opposed to static structure(s), to make inferences on the mechanisms of transport and key interactions.
Neurotransmitter transporters harness the electrochemical potential gradient of ions, namely sodium, across the cell membrane to import neurotransmitters against their electrochemical gradient into neuronal or glial cells. Effective translocation from extracellular (EC)
space regulates neuronal signaling by keeping the levels of neurotransmitters sufficiently low at the synapse. Glutamate is the main excitatory neurotransmitter in the central nervous system. Its high concentrations at the synaptic cleft have been linked to neurological diseases such as epilepsy, stroke, ischemia, and Huntington disease (
). Therefore, glutamate transporters play a key role in preventing excitotoxic effects.
Glutamate transporters belong to the solute carrier 1 (SLC1) family composed of eukaryotic and prokaryotic members that transport acidic or neutral amino acids. The only available high resolution structure for a member of the SLC1 is currently that of the archaeal aspartate transporter from Pyrococcus horikoshii, GltPh. GltPh is a homotrimer. Each monomer is composed of eight transmembrane (TM) helices, TM1–8, and two helical hairpins, HP1 and HP2 (see Fig. 1, A–C), organized in two structural regions: a transport “core” that binds and transports the substrate and sodium ions and a “scaffold” that provides support for the transport core and forms the intersubunit interface. The scaffold is composed of TM1–6, with the trimerization domain formed by TM2, -4, and -5, and the transport core contains the binding pocket that is composed of TM7, TM8, HP1, and HP2. The hairpins reach from opposite sides of the membrane with their tips coming into very close proximity as has been experimentally shown (see Fig. 1D) (
). The most prominent difference between the OF and IF states is the almost rigid body translation of the transport core, together with TM3 and -6, by ∼15 Å into the cytoplasm, accompanied by rigid body rotation of ∼30° in each subunit (see Fig. 1, E and F). This difference is clearly observed upon structural alignment of the trimerization domains in the OF and IF subunits (
). We recently proposed that EAAT1 and/or GltPh would undergo a sequential transition between these two endpoints during the transport cycle, visiting two intermediates composed of 1 (or 2) OF and 2 (or 1) IF subunits (
). Notably, the recently resolved intermediate structure confirmed this prediction. The latter, composed of two IF subunits and one intermediate OF, with the transport domain shifted by ∼3.5 Å and ∼15° toward the IF position (
In all structures resolved in the presence of substrate, the bound aspartate is coordinated by residues on TM7 and -8 and on HP1 and HP2 loops. Additionally, two Na+-binding sites located ∼7 Å from the substrate and from each other have been identified; the first (Na1) is more buried and lies between TM7 and -8, whereas the second (Na2) is located between HP2 and TM7 (5–7) (see Fig. 1, C and D). These structures are in accord with the topology and function of mammalian and prokaryotic glutamate transporters (
Despite this significant progress, much remains to be elucidated with regard to the time-resolved events and inter-residue interactions that mediate sodium-coupled substrate binding or release. For instance, the role of HP2 as an EC gate that controls the binding (or unbinding) of substrate and cations in the OF state has been suggested both by the crystallization of the transporter with an antagonist (
). This role seems plausible because of its high exposure to the EC environment and ability to move therein unobstructed by the rest of the transporter. On the other hand, the IC gating mechanism is far less clear. Crystallographic (
) suggested that HP1 might be involved in IC gating, whereas support from time-resolved examinations at an atomic level has been lacking. Here we conducted a series of molecular dynamics runs, using the high performance computing system, Anton, which permits us to examine processes on the microsecond to millisecond timescale (
) depending on the system size. We were able to view for the first time multiple incidences of IC gate opening and substrate and ion release so as to deduce reproducible patterns and extract statistically reliable information on gating mechanism. The picture that emerged differs from that indirectly inferred from static crystal structures; HP2 (and not HP1) opening is the major event enabling the release of neurotransmitter to the cell interior. HP2 therefore serves as IC gate in the IF state, in addition to its established EC gate role. Our study further highlights the sequence of events that enable the release of substrate, including prior release of Na2 to weaken local interactions and promote substrate dissociation.
Despite the huge amount of data revealed by high resolution crystal structures, the dynamic nature of proteins necessitates the examination of molecular properties beyond those provided by single, static images, such as time-resolved events at the atomic scale. Molecular simulations, empowered in recent years with important advances in computing hardware and software technology such as the Anton supercomputer, serve as a valuable tool to derive detailed information on mechanisms of function, provided that structural data are available (
). Using the structures resolved for GltPh, a prototype for learning about the behavior of EAATs, we were able to delineate the mechanism of substrate unbinding out of the transport core under physiological conditions not necessarily present under the crystallization conditions. The emerging behavior, schematically shown in Fig. 5, is different from that inferred from static structures; a displacement by ∼4 Å at the tip of HP2 loop away from the transport core, or more precisely, an increase by at least 3 Å in the distance between HP2 and TM8 around the space between Pro-356 and Asp-394 is required to initiate the release of neurotransmitter in the IF state, suggesting that HP2 serves as the IC gate. Although the EC gating role of HP2 has been established by several studies, our extensive simulations provide for the first time a concrete visualization of its role as an IC gate in the release of the substrate to the cytoplasm.
It is important to note that the motion of HP2 is required, but may not be sufficient alone to prompt the release of aspartate in the IF state; the increased reorientation and translation ability of the substrate in the transport core succeeding the release of Na2, allowed by an increased separation between HP2 and TM8, and the electrostatic attraction by Arg-276 (neighboring the Ser3 (Ser-277–279) motif at the HP1 tip) all play important roles in driving and completing substrate release. We also note the assisting role of Asp-394 on TM8, which tends to move away from TM7 and HP2 and mediate the interactions of the substrate with Arg-276. The equivalent position of this aspartate in EAAT1 was shown to be essential for substrate interaction with the transporter (
). The crystallized Na2 site and the aspartate pose thus may not necessarily represent the correct or optimal conformation that supports transport. In support of this is the previous experimental observation that in the homologous EAAC1, glutamate in the cytoplasmic binding site dissociates before the three sodium ions (
). The binding pocket shows a tendency to open and release aspartate and sodium at Na2, as shown in this study, rather than remain closed. The closure of the binding pocket and tight binding of the substrate and co-transported ions are prerequisites for the translation of the binding pocket between the external and internal sides of the membrane as the transporter undergoes a global transition from the OF to the IF state. Whether the transition back to an OF state requires the closure of the binding pocket and how anion (chloride) channeling and potassium counter-transport play out in the complete transport cycle are issues that remain to be clarified. In addition, although there is experimental evidence supporting the role of the mammalian equivalent of Asp-405 (in GltPh) at Na1 in cation binding (
), no support for the Na2 site has been reported, mostly due to the proposed coordination of a cation at Na2 by backbone carbonyls, rather than side chain groups that can be mutated to investigate their role in binding or transport.
Therefore, further studies that can ultimately lead to the elucidation of a stable binding pocket conformation that contains an aspartate/glutamate, a proton, and three sodium ions in mammalian transporters, and to the visualization of the other steps of transport cycle including anion transport, are required for fully understanding the mechanism of excitatory amino acid transport from the synapse to the cell interior.
The Anton machine has been provided generously by David E. Shaw Research (
). Anton computer time was provided by the Pittsburgh Supercomputing Center through Grant RC2GM09337. We thank M. Dittrich, N. Simakov, and other staff at the Pittsburgh Supercomputing Center for their support.