Energy Landscape of the Substrate Translocation Equilibrium of

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Energy Landscape of the Substrate Translocation Equilibrium of Plasma-Membrane Glutamate Transporters Jiali Wang, Thomas Albers, and Christof T. Grewer J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b09059 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 11, 2017

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The Journal of Physical Chemistry

Energy Landscape of the Substrate Translocation Equilibrium of Plasma-Membrane Glutamate Transporters

Jiali Wang1, Thomas Albers1,2 and Christof Grewer*,1

1

Department of Chemistry, Binghamton University, Binghamton, NY 13902

2

Current address: Georgia Cancer Center, Augusta University, Augusta, GA 30912

*

To whom the correspondence should be addressed: Christof Grewer, 1Department of Chemistry

Binghamton University, 4400 Vestal Pkwy East, Binghamton, NY 13902, Phone: +1-607-7773250, Fax: +1-607-777-4478, e-mail: [email protected]

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Abstract Glutamate transporters maintain a large glutamate concentration gradient across synaptic membranes and are, thus, critical for functioning of the excitatory synapse.

Mammalian

glutamate transporters concentrate glutamate inside cells through energetic coupling of glutamate flux to the transmembrane concentration gradient of Na+. Structural models based on an archeal homologue, GltPh, suggest an elevator-like carrier mechanism.

However, the energetic

determinants of this carrier-based movement are not well understood. While electrostatics play an important role in governing these energetics, their implication on transport dynamics has not been studied. Here, we combine pre-steady-state kinetic analysis of the translocation equilibrium with electrostatic computations to gain insight into the energetics of the translocation process. Our results show the biphasic nature of translocation, consistent with the existence of an intermediate on the translocation pathway. In the absence of voltage, the equilibrium is shifted to the outward-facing configuration. Electrostatic computations confirm the intermediate state and show that the elevator-like movement is energetically feasible in the presence of bound Na+ ions, while a substrate hopping model is energetically prohibitive. Our results highlight the critical contribution of charge compensation to transport and add to results from previous molecular dynamics simulations for improved understanding of the glutamate translocation process.


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Introduction Plasma-membrane glutamate transport by the members of the SLC1 (solute carrier 1) family (excitatory amino acid transporters, EAATs) is energetically driven by the transmembrane concentration gradient of Na+. Three Na+ ions are transported into the cell for each negativelycharged glutamate molecule together with a co-transported proton1. These ions, when bound to the transporter, move together within the membrane dielectric in the glutamate translocation step. In addition, one K+ ion is counter-transported in a reaction step that is independent of the glutamate translocation step2. In the absence of extracellular and intracellular K+, glutamate transporters are restricted to visit states that are associated with the glutamate translocation steps3. In the additional presence of internal Na+ and glutamate, a homo-exchange equilibrium can be established, in which Na+ and glutamate are exchanged across the membrane in the absence of net flux4-5. When Na+ and glutamate concentration are high enough, in the saturating range, net dissociation to the extraand intracellular solutions can be prevented, allowing isolation of the reaction steps, in which the bound ions are moved across the membrane.

It was previously shown for the glutamate

transporter subtype EAAC1 (excitatory amino acid carrier 16, the rat homologue of the human subtype EAAT37) that this translocation equilibrium is fast, occurring with a time scale of milliseconds, and is associated with transmembrane charge movement, due to the electrogenic nature of the associated reaction steps3, 8. The glutamate translocation process has also been studied in significant detail through x-ray crystallographic structural analysis of the bacterial glutamate transporter homologue GltPh9-10, as well as molecular dynamics (MD) simulations11-13 and anisotropic network models14-15 based on



the structural data.

The current hypothesis is that glutamate translocation is based on an

elevator-type mechanism16, in which the transport domain harboring the bound substrate and Na+ ions moves up and down along the membrane normal, while the trimerization domain remains static, in a fashion similar to an elevator moving up and down the shaft. Strong evidence for this mechanism comes from computational modeling of the inward-facing conformation, based on the inverted repeat structure of the transporter16, as well as from direct crystallographic observation of an inward-facing trimer, which was stabilized by crosslinking10, 17. In addition, one of the structures crystallized with one of the three subunits in a configuration that is close to outward-facing, but with the substrate binding site located slightly deeper in the membrane, suggesting the existence of an intermediate on the translocation pathway18. The translocation reaction of GltPh has also been studied using single molecule spectroscopy of fluorescentlylabeled transporters19. For the mammalian glutamate transporters, less is known about the mechanics and energetics of the glutamate translocation equilibrium.

Furthermore, the energetic contributions of

electrostatics to the translocation pathway are not understood, despite the importance of electrostatics for the electrogenic translocation process20-21. In this work, we have performed pre-steady-state kinetic analysis of the translocation equilibrium in EAAC1, using both voltage jump and concentration jump approaches. The results allow us to determine the microscopic rate constants for two steps involved in the translocation process, suggesting the existence of an intermediate along the translocation pathway. The experimental results are compared with data from kinetic simulations of the translocation equilibrium. We also performed electrostatic calculations based on existing structural models, confirming the existence of an intermediate and providing information of the energy landscape of the 4 ACS Paragon Plus Environment

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translocation equilibrium. While the results are consistent with the elevator-type model, other mechanisms, such as substrate hopping, can be excluded.

Methods Molecular biology.

Rat EAAC1 cDNA constructs3 inserted in the pBK-CMV vector

(Strategene) were transiently transfected into sub-confluent HEK293T (ATCC® Number: CRL 3216) cells, using JetPrime transfection reagent according to the supplier’s protocol (Polyplus). Electrophysiological measurements were performed 1-2 days post-transfection. Electrophysiology and voltage jumps.EAAC1 current recordings were carried out under voltage clamp conditions in the whole-cell recording mode22. The recording pipette resistances were 2-3MΩ, the access resistance 4-6 MΩ. To lock the transporter in translocation equilibrium states, homoexchange conditions were employed5, with saturating concentrations of glutamate and Na+ on both sides of the membrane. The pipette solution contained 120 mM NaMes (Mes = Methanesulfonate), 2mM Mg Mes2, 5mM EGTA (Ethylene glycol-bis(2-aminoethylether)N,N,N',N'-tetraacetic acid), 10 mM HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid), and 10 mM glutamate, adjusted to pH 7.4 with methanesulfonic acid. The extracellular solution contained 140 mM NaMes, 2 mM MgMes2, 2 mM CaMes2, 10 mM HEPES, and 10 mM glutamate also adjusted to pH of 7.4. Voltage jumps (-100 to +80mV) were applied to perturb the electrogenic Na+/glutamate translocation equilibrium, followed by measurement of relaxation currents.


To determine


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EAAC1-specific currents, control currents were recorded in the presence of extracellular 200 µM TBOA (DL-threo-β-benzyloxyaspartic acid) and subtracted from the glutamate-induced currents. For capacitive transient compensation and voltage jump analysis, series resistance compensation of up to 80% was employed. The biphasic decay of the current relaxations were fit with a sum of two exponential functions: I(t) = I1(t = 0) exp(-k1.t) + I2(t = 0) exp(-k2.t)

(1)

here, I1 and I2 are current amplitudes at time t = 0, and k1 and k2 are the relaxation rate constants, respectively. Non-specific transient currents were subtracted in Clampfit (Molecular Devices) and the EAAC1 specific component fit to a Boltzmann-like function: Q(Vm) = Qmin + Qmax· [1 + exp(ZQ(VQ-Vm)· F/(RT))]−1)

(2)

Here, Q represents the charge moved, ZQ the valence of this charge, and VQ the midpoint potential of the charge movement. R is the gas constant, T the temperature, and F the Faraday constant. Time constants for relaxation current decay were determined using single- or double-exponential fits in pClamp 9 software (Molecular Devices). Computation of the electrostatic energy of the transporter. We used the Adaptive PoissonBoltzmann Solver, APBS

23

, together with the APBSmem Java routines

24

to calculate

electrostatic energies of the glutamate transporter conformers as a function of the translocation reaction coordinate generated by targeted and steered molecular dynamics simulations, see below. A modified version of the linearized Poisson-Boltzmann equation (LPBE) was used 25, as detailed in 20: 6 ACS Paragon Plus Environment

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2

− ∇[ε (r )∇φ (r )] + κ (r )φ (r ) =

e4π ρ (r ) k bT

(3)

Here, ε is the dielectric constant, which depends on the spatial coordinate r. φ is defined as eΦ/kbT, in which Φ is the electrostatic potential and e the elementary charge, T the temperature, and kb the Boltzmann constant. κ is the Debye-Hückel screening constant, and ρ is the charge density. The dimension of the box at the finest focusing was 130 Å cubed with 161 grid points per cube in each dimension. With the potential maps, the total electrostatic energy, E, was then computed, integrating over the product of the charge and potential as a function of the spatial coordinate26:

E = ∫ φ (r )ρ (r )dV Here, dV is a volume element.

(4)

To compute the valence of charge movement induced by

transporter conformational changes, membrane potentials of varying magnitude, V, were applied to the internal side of the membrane, introducing an additional term in eq. (3): 2  κV e4π  ρ (r ) + − ∇[ε (r )∇φ (r )] + κ (r )φ (r ) = f (r )   kbT  4π  2

(5)

Subsequently, the difference in total electrostatic energy, ∆E, for two protein configurations was calculated and the valence was obtained from the slope of the ∆E vs. V relationship27. Energy contributions that do not come from movement of protein charges, i.e. reshaping of the electric field, have been subtracted in this approach, after calculating the electrostatic energy in the absence of protein charges. Protonation states of ionizable groups in the membrane (see conserved charged residues described in section "minimal model") was set to their protonation 7 ACS Paragon Plus Environment


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state in aqueous solution, except for E373, which is predicted to be protonated in the fully-loaded complex 28.

Molecular dynamics simulations. The model system for MD simulations was generated with VMD software 29, using GltPh (PDB code 2NWX 9) or an EAAT3 homology model based on the GltPh structure. In the EAAT3 homology model, two or three co-transported Na+ ions and one aspartate molecule were bound to each subunit as previously reported21, and E373, as the potential proton acceptor28, was protonated. Sequence alignment was performed with ClustalW and the homology model was built with the Modeller program30. The GltPh structure or the EAAT3 homology model were inserted in a pre-equilibrated POPC lipid bilayer with the dimensions 130 x 130 Å. TIP3P water was added to generate a box measuring 100 Å in the zdirection. The minimum distance between periodic images was 41 Å in the x and y directions (plane of the membrane), and 42 Å in the z-direction. NaCl was added at a concentration of 150 mM and the system was neutralized. The total number of atoms in the system were 146891 (GltPh) and 146859 (EAAT3). Simulations were performed using NAMD 2.8b CHARMM27 force field

32

31

using the

, after several minimization and equilibration steps. The cutoff for

short-range interactions was 12 Å. particle-mesh Ewald method

33

For long-range electrostatic interactions, we used the

implemented in NAMD. Bonds to hydrogen atoms and TIP3P

water were kept rigid using SHAKE. The time steps of the simulations were 2 fs. The complete simulation system including substrate, lipid and water is shown in Supplementary Fig. S1. Intermediate structural models were generated using targeted MD (TMD) with the outwardfacing structure as the starting and the inward-facing GltPh structure, 3KBC10 as the end point of the simulation (two runs, the RMSD for the intermediate was 2.65 Å). The RMSD between beginning and end structures was 9.96 Å. For electrostatic calculations, 10 to 20 frames were 8 ACS Paragon Plus Environment

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used in equidistant time points along the time axis from fully outward to fully inward facing state. The length of the TMD simulations was 10 ns. The spring constant was 250 kcal/mol/Å2. Electrostatic calculations using APBS were performed on single conformers at equidistant time points along the trajectory. For steered MD (SMD) simulations, a constant velocity pulling protocol (5 Å/ns) was used with the Cα atom of the bound aspartate substrate as the point of force application. The spring constant was 7 kcal/mol/Å2 for the spring between dummy atom and aspartate. The Force was applied in the z direction, along the membrane normal (illustrated in Supplementary Fig. S1). To ensure that the applied force did not move the entire transporter within the simulation box, the C(α) atoms of the N-terminal valines in each subunit were restrained with a harmonic potential (k = 1 kcal/mol/Å2). This steering protocol resulted in a rigid body movement of the whole transport domain, in the absence of substrate dissociation from its binding site, in both the forward and reverse direction (RMSD of 2.7 Å). SMD simulation lengths were chosen as either 8 or 16 ns. The SMD simulations in forward direction were performed in triplicate for both restrained and un-restrained conditions. The elevator-like rigid body motion of the transport domain was observed for all three simulations. To model substrate hopping, we weakly restrained the backbone C(α) atoms in the transport domain with a harmonic potential with a force constant of k = 0.01 kcal/mol/Å2, in order to prevent global motion of this domain in an elevator-like fashion along the membrane normal, but to allow lateral motion of the substrate along the translocation pathway in a hopping-like manner. Force was applied to the C(α) atom of aspartate in the z direction, along the membrane



normal (illustrated in Supplementary Fig. S1). The pulling rate was 10 Å/ns in the z-direction of the membrane.

Minimal electrostatic model. In this model, only 7 conserved charged residues were included (illustrated in Supplementary Fig. S2), with positions from the EAAT3 homology model for the outward and inward-facing conformations (based on PDB structures 2NWX and 3KBC10). These positions were: D367, E373, D439, D443, R444, R446, D454. The charges were modeled as point charges. The bowl was modeled as a cone using APBSmem24 with an inner exclusion radius of 0 Å and an outer exclusion radius of 20 Å. The dielectric constants were 2 or 10 for the membrane and 80 for the solvent.

Results Charge movement associated with substrate translocation relaxes with biphasic kinetics. To determine the kinetics of the substrate translocation process, we applied experimental conditions, in which the transporter is restricted to visit states along the translocation pathway (principle of the method shown in Fig. 1A). For this purpose, saturating concentrations of Na+ and substrate (glutamate or aspartate) were applied on both sides of the membrane, facilitating the exchange reaction, but largely preventing substrate/Na+ dissociation to the extracellular or intracellular side5. Subsequently, the electrogenic exchange/translocation equilibrium20-21 was disturbed using a step in the membrane potential, followed by observation of the current relaxation until the new equilibrium is reached (Fig. 1). As illustrated in Fig. 1D, a voltage jump from +80 mV to -60 mV (voltage protocol shown in Fig. 1B) resulted in a rapidly-rising transient current, which decayed to the baseline after 30-40 ms, at which point equilibrium is reached and net flow between states ceases. The relaxation of the current could not be fit with a single-


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exponential function, but required a sum of two exponential terms for adequate fit (Fig. 1D, residuals shown in C). The biphasic decay of the current indicates that more than one step is associated with the substrate translocation equilibrium.

Location of the substrate exchange equilibrium depends on the membrane potential. The voltage dependence of the exchange equilibrium was tested by first clamping the voltage to +80 mV, to push the transporter mainly to the outward-facing state, followed by negative voltage steps to potentials of up to -100 mV (Fig. 2A), forcing re-equilibration of the electrogenic process to a more inward-facing configuration. As shown in Fig. 2B, both the transient current amplitude, as well as the time constant of the relaxation were dependent on the transmembrane potential. The experimental data were fit with a kinetic model that includes an intermediate along the translocation pathway (Fig. 1A), neglecting substrate/Na+ dissociation steps. The results of this fit are illustrated in Fig. 2C.

They are consistent with two consecutive

translocation steps that have very similar valence, but one step equilibrating about 5 times faster than the other. The values for the rate constants of the forward and reverse reactions for each step are shown in the legend of Fig. 2. To analyze the voltage dependence of the charge movement associated with the two-step electrogenic translocation process, we integrated the transient current signals. As shown in Fig. 2D, the translocated charge increased with increasing hyperpolarization of the membrane, following a Boltzmann-like behavior for both phases of the current relaxation. This behavior is expected based on the voltage-dependent redistribution of the translocation equilibrium, in which charge movement saturates when the transporter is fully outward- or inward facing, at extreme positive and negative potentials, respectively.



The decay rate of the charge movement depends on the transported substrate. If the voltage jump-induced charge movement represents the translocation reaction, the kinetics of the relaxation should be influenced by the nature of the transported substrate. For example, Daspartate, which is transported at a lower steady-state rate compared to L-glutamate34, should induce transient currents that relax with slower kinetics. Consistent with this expectation, the relaxation rate constants, 1/τ, for both fast and slow phases were significantly slower in the presence of D-aspartate compared with L-glutamate (Fig. 3), suggesting that the current relaxations reflect substrate-dependent events in the translocation process. In contrast, cysteine, which is also a transported substrate4, elicited currents with faster decay rate constant than glutamate for the slow phase, but slower decay of the fast phase (Fig. 3). These results indicate that the decay rates of the transient current are representative of substrate-dependent reactions steps, most likely the translocation processes.

Decay rate constants are consistent with a voltage dependent two-step equilibration process. For a reversible process, the observed rate constant for the relaxation should be a sum of the rate constants for the forward, kf, and reverse reaction, kr. If the reversible process is also voltage dependent, the rate constants for forward and reverse reactions will depend inversely on the membrane potential35, as proposed for transport processes by Läuger36. Consistent with this analysis, the observed relaxation constants for the slow and the fast phase of the transient current were dependent on the membrane potential, increasing at both negative and positive potentials. Because translocation is associated with the movement of positive charge, the rate constant for the reverse reaction dominates at positive potentials, whereas the forward reaction dominates at negative potentials. The relationship of the observed rate constant, kobs, and voltage, V, can be represented by the following equation, 12 ACS Paragon Plus Environment

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= + = (0) ⁄ + (0) ⁄

(6)

assuming a symmetric barrier, with kf(0) and kr(0) being the rate constants for forward and reverse reaction at V = 0 mV, respectively. z is the valence of the process and R, F and T have their usual meaning. The solid lines in Fig. 4 show fits to this equation, representing the experimental data well.

A channel-like substrate hopping model is not consistent with the experimental data. In a previous report, steered molecular dynamics (SMD) simulations, in which a force was applied to the substrate, effectively pulling it in the z-direction lateral to the membrane, was used to accelerate the translocation process, suggesting a potential translocation pathway13. In this previous study, the substrate moved between semi-stable positions along the membrane lateral without a major conformational change of the transporter in a substrate hopping-like pathway13 (illustrated in Fig. 5B, the transport domain is visualized in Fig. 5A). When we performed analogous simulations with an identical pulling rate of 10 Å/ns on the C(α) position of the aspartate substrate, we did not observe substrate hopping, but rather elevator-like movements of the whole transport domain along the z-axis, with bound aspartate remaining stable in the substrate binding site (see below, next section). We then weakly restrained the C(α) atoms in the transport domain, using a harmonic potential, to prevent this domain from moving in an elevator like fashion, while allowing the restrained atoms enough motion to permit the substrate to be pulled along the membrane normal. Under these conditions and at the same pulling rates, aspartate moved in the z-direction, hopping between three major positions, about 20%, 40% and 80% of the full translocation pathway, while the fold



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of the transport domain remained essentially static (no elevator-like motion). Similar results were observed in three independent simulations. Next, we determined the electrostatic energy associated with the substrate hopping mechanism by solving the Poisson-Boltzmann equation along the translocation pathway (Fig. 5).

The

electrostatic energy, ∆Gelec, as a function of the displacement of aspartate along the z-axis, as well as transport protein displacement along the membrane normal is shown in Fig. 5C. Clearly, the positioning of GltPh in the membrane at 15 Å distance from the bottom of the membrane is energetically the most favorable. The energy profile along the reaction coordinate at the ideal insertion depth of the transporter is shown in Fig. 5D. While at least one intermediate is observed along the translocation pathway, consistent with results from Bahar et al.13, the energy necessary to overcome the electrostatic barrier of about 300 kJ/mol appears to be physically unrealistic. In addition, we calculated the charge displacement during substrate hopping, by applying an electric field during the electrostatic calculations (Fig. 5E). The valence, which was calculated according to eqns. (4) and (5), becomes fully negative in the second half of the translocation coordinate, but is positive before the intermediate is reached (Fig. 5E). This behavior is not consistent with the experimental data, which show positive valence for both phases of the translocation process.

Together with the large electrostatic energy barrier calculated for

translocation, we conclude that the substrate hopping model is not physically plausible.

An elevator-like transport mechanism is consistent with the experimental data and predicts an intermediate on the translocation pathway.

The next mechanism we evaluated in

comparison to experimental data was the elevator-like transport mechanism, which was


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postulated in the literature16,

37-38

, as conceptually illustrated in Fig. 6A. Here, the whole C-

terminal transport domain translocates from the outward-facing to the inward-facing configuration in an elevator-like fashion (see arrow in Fig. 6A). Transitions between outwardand inward-facing states of GltPh were modeled using a targeted MD (TMD) protocol, with the target structure based on the 3KBC crystal structure10. Similar results were obtained using SMD simulations, in which the aspartate molecule was pulled at the C(α) atom in the z-direction (Supplementary Fig. S1, arrow), without restraining atoms in the transport domain of the protein.

In these simulations, the transport domain

translocated in a rigid-body, elevator-like motion from the outward to the inward facing state, with the aspartate remaining in the substrate binding site. Upon reaching the inward-facing state, aspartate dissociated through partial opening of hairpin loop 1 (HP1). This dissociation part of the trajectory was not included in the electrostatic analysis. Next, we calculated the electrostatic energy, ∆Gelec, as a function of the reaction progress along the translocation coordinate. As shown in Fig. 6B, an intermediate with low electrostatic energy was identified at 35% translocation progress. The barriers associated with the reaction are in the range of 60 kJ/mol for the configuration bound to one aspartate and two sodium ions, indicating physically feasible energy barriers for translocation. In contrast, the electrostatic energy barrier for relocation of the apo-state is 185 kJ/mol, indicating a much slower relocation process. The energy landscapes for both the Na2Asp-bound and the apo-state are shown in Figs. 6C and D, showing the lowest electrostatic energy barrier near the -10 Å insertion depth. Interestingly, the inward-facing state has a higher electrostatic energy than the outward-facing state (Fig. 6B), suggesting electrostatic stabilization of the outward-facing transporter conformation, and



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preference of this state at equilibrium. It should be noted that the electrostatic energy is a very important term, but only one of several other terms contributing to the optimal insertion depth of the transporter in the membrane. As expected the energy barrier depends on the dielectric constant of the protein, εP, used for the calculations (Fig. 6E). With a εP of 2.0, the energy barrier is about 4-times higher than with εP = 10. Finally, we determined the valence of the charge movement along the translocation pathway. As shown in Fig. 6F, the valence is positive along the reaction pathway for configurations with aspartate and 2 or 3 Na+ ions bound. This result is in agreement with the experimental data, showing positive valence for formation, as well as decay of the intermediate. In contrast, the valence of the relocation of the apo-form of the carrier is negative, due to the movement of the negatively-charged ion binding sites through the membrane electric field, as predicted previously20. It should be noted that the targeted MD approach may lead to an intermediate that may be structurally slightly different than the real intermediate.

However, since the lower-level

electrostatic calculations are not performed at atom-level resolutions (0.8 Å grid spacing), small deviations from the real structure of the intermediate are not expected to have a major effect on the results. In addition, pulling rates of < 10 Å/ns had little effect on the trajectory of the outward-to-inward facing transition.

Thus, deviations of electrostatic energies from actual

values for the transition pathway cannot be excluded, but should be minor. The predicted intermediate along the translocation pathway, together with the outward- and inward-facing configurations, are shown in Figs. 7B, D and F. The iso-potential lines calculated after applying a membrane potential of -100 mV to the inside of the membrane are shown in


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Figs. 7A, C and E.

As proposed previously

20

, the transporter structure distorts the

transmembrane electric field compared to the linear potential drop in a pure membrane, resulting in a defocusing of the electric field.

While progressing along the translocation reaction

coordinate for the outward-inward-facing transition, the iso-potential lines are progressively shifted towards the cytoplasm. These results suggest that, at physiological membrane potentials, part of the free energy change of the translocation process, ∆Gelec, is contributed by reshaping of the membrane electric field by the conformational change.

A minimal structural model reveals the important conserved charges. Several charged amino acid residues are conserved in the SLC1 acidic amino acid transporter family, as well as GltPh. We have generated a minimal structural model containing only these conserved charges, as well as the transported substrate and Na+ ions in an implicit membrane environment (Fig. 8A). Subsequently, we performed Poisson-Boltzmann analysis of the electrostatic energy as a function of the translocation progress. The results (Fig. 8B-D) demonstrate that the barrier along the translocation coordinate is in the same range as for the full protein model, demonstrating that the essence of the physics of the process can be reproduced with dramatically simplified assumptions. Interestingly, inclusion of an extracellular bowl in the membrane dielectric, as evident in the GltPh structures, reduces the electrostatic energy barrier (Figs. 7B-D), indicating that this bowl may contribute to the fine balance of energetic components to the translocation process.

Translocation energetics in an EAAT3 homology model. We had previously generated a homology model of EAAT3 based on the GltPh structure21. This homology model was used to calculate ∆Gelec along the translocation reaction coordinate. As shown in Fig. 9, the electrostatic



energy hypersurface reproduces the general features observed for GltPh, including the favorable energy of the outward-facing state over the inward-facing state, as well as intermediate(s) along the translocation pathway. It should be noted that, in contrast to GltPh, we were unable to induce the outward- to inward-facing transition in EAAT3 through steered MD by applying force in zdirection to the substrate alone, which resulted in dissociation of the substrate (aspartate) from the binding site. This differential behavior may be caused by the higher affinity of GltPh for aspartate39 compared to EAAT33. Therefore, to induce the elevator-like translocation process, force was also applied to the Cα atom R446, a residue which coordinates the β-carboxylate of aspartate.

Discussion This work aimed at the identification of energetic contributions to the conformational changes associated with substrate translocation by the plasma membrane glutamate transporters, including the mammalian transporter EAAC1 and the archeal homologue GltPh. In particular, the work focused on the contributions of the electrostatic energy, due to the variety of charged species that are co-transported with glutamate, as well as the charged amino acid side chains moving within the electric field of the membrane20.

Therefore, it is expected that the

electrostatic energy contributes in a major way to the energy barrier along the translocation pathway. The major findings of our work are that 1) electrostatic energy barriers are in the 50 kJ/mol range for the fully-loaded carrier, consistent with rapid translocation kinetics. 2) The electrostatic energy and valence profile is consistent with an elevator-like carrier model, rather than a substrate hopping mechanism. 3) The energy landscape along the translocation pathway shows an intermediate, consistent with kinetic data with two exponential phases of the relaxation


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of the translocation equilibrium. 4) A translocation equilibrium that energetically favors the outward-facing configuration over the inward-facing state. Our results highlight the importance of electrostatic contributions to the translocation process, and underline the charge compensation mechanism, which was proposed previously20. In this mechanism, positive charge of the co-transported cations is partially compensated by negative charge of the cation binding sites, thus reducing the overall charge of the substrate/cation/carrier complex that moves across the membrane. Consistently, the fully-loaded transporter undergoes translocation with much lower energy barrier than the apo form of the transporter (Fig. 6), explaining why the relocation is the rate limiting step in the transport cycle3, 40.

Intermediate and translocation pathway. The biphasic decay of the transient transport current in exchange mode provides strong evidence for two sequential steps associated with the translocation equilibrium, requiring an intermediate along the translocation pathway.

The

kinetics of decay are not consistent with parallel reactions, which should show singleexponential decay characteristics. However, an alternative explanation could be the existence of two transporter populations with divergent translocation kinetics. This scenario is less likely because the relative amplitude of both phases did not vary with experimental parameters such as transporter expression level or time after transfection, both of which may affect relative levels of two independent transporter populations. Evidence for an intermediate on the translocation pathway has been presented previously in the literature, for example through MD simulations12.

In addition, a structure of GltPh was

published, in which one subunit of the homotrimer deviates from the outward-facing configuration18. The intermediate proposed in our TMD/SMD simulations on the basis of the



electrostatic energy is located at about 35%-40% of the overall translocation progress from full outward-facing to full inward facing. Electrostatic valences for transition to and from this predicted intermediate (0.21 and 0.32, Fig. 6F) are in the same range as those from the experimental data (0.35 and 0.4, Fig.2). Thus, this predicted intermediate may provide a starting point for future structure function and computational analysis. The GltPh structure, pdb code 3V8G, in comparison, is much closer to the outward-facing state18. Adding to these previous reports, our experimental and computational data suggest that the two-step translocation process is a general feature of both archeal and mammalian glutamate transporters and, thus, most likely a conserved mechanistic feature of the SLC1 superfamily.

Location of translocation equilibrium.

The translocation equilibrium associated with

transition from the outward- to the inward-facing states has been subject to studies in the past17, 41

. The fact that GltPh crystallizes in the outward-facing state, while the inward-facing state

needs to be stabilized by crosslinking10, points to the translocation equilibrium shifted toward the outward-facing configuration. configuration.

However, crystal contacts may favor one over the other

More direct evidence was recently published with data from atomic force

microscopy (AFM) on GltPh. Interestingly, “up” and “down” states were directly observable in AFM imaging17. Dwell times indicate that the “up” (outward-facing) state is occupied for longer times, suggesting a stabilization of about 3.5 kJ/mol relative to the inward-facing state17. Our kinetic data on mammalian EAAC1 suggest that the outward-facing state is also more stable in this mammalian transporter. Analysis of the rate constants of the relaxation of the translocation equilibrium suggest that the transporter spends 44% of the time in the outward-facing state, vs. 23% in the inward-facing state, compatible with a stabilization of about 1.6 kJ/mol of the outward-facing configuration.

Interestingly, our electrostatic calculations also suggest 20 ACS Paragon Plus Environment

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stabilization of the outward-facing state, but to a much higher degree, with an energy difference of about 40 kJ/mol (Fig. 6B). Therefore, it can be speculated that while electrostatic energy is a major contributor to the stabilization of the outward-facing state for both GltPh and EAAC1, other factors must play a role in the weak preference for the outward-facing state.

Energy barriers along the translocation pathway.

Our Poisson-Boltzmann calculations

suggest an energy barrier in the range of 60 kJ/mol for the electrostatic component of the energy. This value can be compared with activation energies previously determined for the fast and slow phase of the transient current decay associated with translocation. These activation energies were in the 100-120 kJ/mol range.

Thus, it is likely that other factors contribute to the

translocation barrier (see below). These values of activation energies are compatible with values previously published for glutamate transporters, as well as other sodium coupled transporters, and allow rapid translocation of substrate across the membrane. In contrast, the Born energy for inserting only one Na+ ion into the membrane (ε = 2) would be 350 kJ/mol making translocation prohibitive in the absence of charge compensation mechanisms provided by the transporter binding sites.

Substrate hopping model. A previous MD study investigated a substrate hopping mechanism of aspartate moving along a channel, by applying a steering force to the aspartate molecule13. In our hands, applying inward force to the Cα atom of the aspartate ligand always resulted in rigid body movement of the whole transport domain in elevator-like fashion, without dissociation of aspartate from its binding site. To achieve aspartate hopping in a channel-like fashion, we had to restrain backbone carbon atoms in the transport domain, using a harmonic potential. The reasons for this discrepancy between our results and the previous study13are not known. The energetic



barrier associated with the substrate hopping mechanism are, however, inconsistent with experimental data and suggest that this mechanism is not energetically feasible. This finding supports a number of other reports based on structural and spectroscopic data that propose the elevator-type model10, 17, 19 and that are not consistent with substrate hopping.

Contributions to energetics of structural changes. Previous results obtained from transporters with charge-altering mutations suggest that the electrostatic energy is of prime importance in controlling the energy landscape along the translocation pathway28, 42-44, although some of these mutations also alter substrate/cation binding and, thus, make interpretation of results less straightforward. In addition to electrostatics, several other factors are expected to contribute to the energy barrier and, thus, the kinetics of the translocation process. One of these factors is the apolar energetic term45.

Analysis of the structure of the GltPh transporter has indicated

significant hydrophobic interaction at the interface between the transport domain and the trimerization domain10, facilitating the elevator-like motion of the transport domain along the membrane normal.

Further experimentation/computations will be necessary to assess the

magnitude of the contribution of this apolar effect, although it is expected that it is significant. In addition, specific polar18 and hydrogen bonding interactions are broken and formed during the translocation process. Such polar interactions in a hydrophobic background may also contribute energetically to the barrier and have previously been shown to be important in protein-protein interaction sites46. Finally, it has been suggested that dissociation of the transport domain from the trimerization domain is necessary for translocation to occur18. This may be associated with increased water permeability of either domain, raising the possibility of solvent and entropic effects.

Based on our results of the much larger computed electrostatic energy difference

between inward- and outward-facing states compared to the experimental results suggest that 22 ACS Paragon Plus Environment

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these other contributions may, in fact, be important for keeping the translocation equilibrium in balance, without a dramatic favoring of one of the two alternating access states, as based on electrostatics.

Conclusions. To summarize, our results underline the importance of electrostatic contributions to the energetics of the glutamate translocation process, as well as the validity of the elevator-like mechanism that was predicted based on the static end-state structures of outward- and inwardfacing configurations.

A balance between negative charge of the cation binding sites and

positive charge of the translocated ions is required to lower the activation barrier associated with the elevator-like mechanism.

In addition, the electrostatic energy landscape predicts an

intermediate along the translocation pathway, consistent with experimental data showing biphasic nature of the relaxation of the translocation equilibrium.

In contrast, a substrate

hopping model is not consistent with the experimental data and energetically prohibitive. Thus, it is important to include the electrostatic energy contribution in future structural and kinetic models of glutamate transporter function.

Acknowledgements This work was supported by by the National Science Foundation Grant 1515028 awarded to CG.

Supplementary Information The Supporting Information is available free of charge on the ACS Publications website. Illustration of the MD simulation box and of the location of conserved charges in the EAAC1 transport domain.



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Figure Legends Figure 1: Current relaxations in substrate exchange mode display biphasic kinetics. (A) Kinetic scheme of the substrate exchange mode with saturating concentrations of Na+ and glutamate on both sides of the membrane. The predominant reaction is equilibration of the translocation step(s), bold grey arrow.

The potential existence of an intermediate on the

translocation pathway is indicated by the (TGluNa3) state. T represents the transporter, Glu is glutamate, and Na are the sodium ions.

(B) Voltage protocol used for the voltage jump

experiments. (C) Residuals of a single-exponential fit (blue) and double-exponential fit (red) to the experimental data shown in (D). (D) Current relaxation in response to the voltage jump. The substrate concentration was 10 mM, with 140 mM NaMes on both sides of the membrane.

Figure 2: Voltage dependence of the exchange current. (A) Voltage protocol used for the voltage jump experiments. (B) Current relaxations in response to voltage jumps. The substrate concentration was 10 mM, with 140 mM NaMes on both sides of the membrane. (C) Fit of the simplified kinetic model shown in (D) to the original data. The rate coefficients were: k1 = 0.7 ms-1, k-1 = 1 ms-1, k2 = 0.15 ms-1, k-2 = 0.2 ms-1,z1 = 0.35, z2 = 0.4. The data were fitted using a global fit based on numerical integration of the rate equations pertaining to the kinetic scheme in Fig. 2D: I(t,V) = Const..(k1(V) Tout - k-1(V) Tinter) + (k2(V) Tinter - k-2(V) Tin)

(7)

with ki(V) = ki(V = 0) exp(±ziFV/(2RT))

(8)

where the index i denotes the particular rate constant in Fig. 2D.


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The valences were obtained from the Boltzmann relationship in Fig. 2E and fixed. The other four parameters (forward and reverse rate constants) were left to adjust by the fitting routine. The relaxation rates of the slow and the fast phase are governed by the sum of the rate constants for the forward and backward reactions. These two parameters are interdependent when analyzing the reaction rate alone, but they become parametrized through the current amplitudes. Because of the global fitting routine, we do not feel confident to give exact error intervals for the rate constants, but we assume an error of ±30%. (E) Voltage dependence of the charge movement obtained by integrating the transient currents in (B) over time. The solid lines are fits to a Boltzmann relationship. The valences, zi, were 0.5 ± 0.1 for the fast phase and 0.46 ± 0.4 for the slow phase.

Figure 3: Substrate dependence of relaxation rate constant. The relaxation rate constants for the fast (grey bars) and slow phases (black bard) of the homo-exchange current decay were determined for the substrates L-glutamate, L-cysteine, and D-aspartate at a voltage of V = 0 mV and a substrate concentration of 1 mM at both sides of the membrane. The [Na+] was 140 mM. The stars indicate significance based on Student’s t-test analysis relative to the rate constants for glutamate exchange.

Figure 4: Voltage dependence of relaxation rate constants. The relaxation rate constants, 1/τ, for the fast (open circles) and slow phase (closed circles) of the homo-exchange current decay were determined as a function of the membrane potential in the presence of 1 mM glutamate on both sides of the membrane. The [Na+] was 140 mM. The solid lines represent fits to eq. 6. The fit parameters were: Slow phase: kf(0) = (0.06 ± 0.01) ms-1, kr(0) = (0.06 ± 0.01) ms-1, z = 0.8 ± 0.7. Fast phase: kf(0) = (0.65 ± 0.40) ms-1, kr(0) = (1.15 ± 0.55) ms-1, z = 0.42 ± 0.29.



Figure 5: A substrate hopping is inconsistent with the experimental data and results in a large energy barrier. All following panels are for GltPh. (A) Illustration of one subunit of the GltPh trimer. The transport domain is shown in red and the bound substrate (aspartate) in space fill. (B) Illustration of the movement of substrate through the transport domain in a substrate hopping mechanism. (C) ∆Gelec hypersurfaces in the Na2Asp-bound state. The y-axis represents the insertion level of the transporter in the membrane dielectric. (D) Minimum pathway polar (electrostatic) solvation energy as a function of the reaction coordinate. (E) Computed valence along the reaction coordinate. ∆Gelec was calculated by solving the linearized Poisson-Boltzmann equation using the APBS routine.

Figure 6: The elevator-like carrier model predicts the existence of an intermediate on the translocation pathway. All following panels are for GltPh. The elevator-like transition of one of the three subunits used for the electrostatic calculations is illustrated in (A). (B) Polar (electrostatic) solvation energy as a function of the reaction coordinate. (C, D) ∆Gelec hypersurfaces in the Na2Asp-bound and apo states. The y-axis represents the insertion level of the transporter in the membrane dielectric. (E) Maximum barrier height for transition between several transporter states. (F) Computed valence along the reaction coordinate. ∆Gelec was calculated by solving the linearized Poisson-Boltzmann equation using the APBS routine.

Figure 7: Reshaping of the electric field during the outward- to inward-facing transition. Electrostatic calculations of the iso-potential lines (A, C, E) were performed using APBSmem with an internal potential -100 mV and an external potential of 0 mV, after setting all protein charges to 0. Structures were obtained through steered MD (B, D, F). The intermediate structure represents the translocation progress at the energy minimum, Fig. 6B, at about 35% completion of translocation. 32 ACS Paragon Plus Environment

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Figure 8: A minimal transport model with charge compensation and extracellular bowl can explain the data. (A) Illustration of the model with extracellular bowl with negative charges shown in red and positive charges shown in blue. The wire mesh indicates the transition from ε = 2 for the membrane and ε = 80 for the solvent. (B) Polar (electrostatic) solvation energy as a function of the reaction coordinate.(C, D) ∆Gelec hypersurfaces in the fully loaded state in the presence and absence of the bowl. The y-axis represents the insertion level of the transporter in the membrane dielectric.

Figure 9:

Electrostatic energy contribution to translocation in EAAT3.

∆Gelec

hypersurfaces in the Na3Asp-bound state. The y-axis represents the insertion level of the transporter in the membrane dielectric. The transition was modeled using a EAAT3 homology model based on the GltPh structure and steered MD, applying force to the Cα atoms of the substrate and R446.



TOC graphic


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Figure 1: Current relaxations in substrate exchange mode display biphasic kinetics. (A) Kinetic scheme of the substrate exchange mode with saturating concentrations of Na+ and glutamate on both sides of the membrane. The predominant reaction is equilibration of the translocation step(s), bold grey arrow. The potential existence of an intermediate on the translocation pathway is indicated by the (TGluNa3) state. (B) Voltage protocol used for the voltage jump experiments. (C) Residuals of a single-exponential fit (blue) and double-exponential fit (red) to the experimental data shown in (D). (D) Current relaxation in response to the voltage jump. The substrate concentration was 10 mM, with 140 mM NaMes on both sides of the membrane. 134x204mm (300 x 300 DPI)

ACS Paragon Plus Environment


Figure 2: Voltage dependence of the exchange current. (A) Voltage protocol used for the voltage jump experiments. (B) Figure 2: Voltage dependence of the exchange current. (A) Voltage protocol used for the voltage jump experiments. (B) Current relaxations in response to voltage jumps. The substrate concentration was 10 mM, with 140 mM NaMes on both sides of the membrane. (C) Fit of the simplified kinetic model shown in (D) to the original data. The rate coefficients were: k1 = 0.7 ms-1, k-1 = 1 ms-1, k2 = 0.15 ms-1, k-2 = 0.2 ms-1, z1 = 0.35, z2 = 0.4. The data were fitted using a global fit based on numerical integration of the rate equations pertaining to the kinetic scheme in Fig. 2D: I(t,V) = Const..(k1(V) Tout - k1(V) Tinter) + (k2(V) Tinter - k-2(V) Tin) (7) with ki(V) = ki(V = 0) exp(±ziFV/(2RT)) (8) where the index i denotes the particular rate constant in Fig. 2D. The valences were obtained from the Boltzmann relationship in Fig. 2E and fixed. The other four parameters (forward and reverse rate constants) were left to adjust by the fitting routine. The relaxation rates of the slow and the fast phase are governed by the sum of the rate constants for the forward and backward reactions. These two parameters are interdependent when analyzing the reaction rate alone, but they


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become parametrized through the current amplitudes. Because of the global fitting routine, we do not feel confident to give exact error intervals for the rate constants, but we assume an error of ±30%. (E) Voltage dependence of the charge movement obtained by integrating the transient currents in (B) over time. The solid lines are fits to a Boltzmann relationship. The valences, zi, were 0.5 ± 0.1 for the fast phase and 0.46 ± 0.4 for the slow phase. 202x529mm (300 x 300 DPI)



Figure 3: Substrate dependence of relaxation rate constant. The relaxation rate constants for the fast (grey bars) and slow phases (black bard) of the homo-exchange current decay were determined for the substrates L-glutamate, L-cysteine, and D-aspartate at a voltage of V = 0 mV and a substrate concentration of 1 mM at both sides of the membrane. The [Na+] was 140 mM. The stars indicate significance based on Student’s ttest analysis relative to the rate constants for glutamate exchange. 50x45mm (300 x 300 DPI)


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Figure 4: Voltage dependence of relaxation rate constants. The relaxation rate constants, 1/τ, for the fast (open circles) and slow phase (closed circles) of the homo-exchange current decay were determined as a function of the membrane potential in the presence of 1 mM glutamate on both sides of the membrane. The [Na+] was 140 mM. The solid lines represent fits to eq. 4. The fit parameters were: Slow phase: kf(0) = (0.06 ± 0.01) ms-1, kr(0) = (0.06 ± 0.01) ms-1, z = 0.8 ± 0.7. Fast phase: kf(0) = (0.65 ± 0.40) ms-1, kr(0) = (1.15 ± 0.55) ms-1, z = 0.42 ± 0.29. 88x72mm (300 x 300 DPI)



Figure 5: A substrate hopping is inconsistent with the experimental data and results in a large energy barrier. All following panels are for GltPh. (A) Illustration of one subunit of the GltPh trimer. The transport domain is shown in red and the bound substrate (aspartate) in space fill. (B) Illustration of the movement of substrate through the transport domain in a substrate hopping mechanism. (C) ∆Gelec hypersurfaces in the Na2Asp-bound state. The y-axis represents the insertion level of the transporter in the membrane dielectric. (D) Minimum pathway polar (electrostatic) solvation energy as a function of the reaction coordinate. (E) Computed valence along the reaction coordinate. ∆Gelec was calculated by solving the linearized Poisson-Boltzmann equation using the APBS routine. 270x353mm (300 x 300 DPI)


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Figure 6: The elevator-like carrier model predicts the existence of an intermediate on the translocation pathway. All following panels are for GltPh. The elevator-like transition of one of the three subunits used for the electrostatic calculations is illustrated in (A). (B) Polar (electrostatic) solvation energy as a function of the reaction coordinate. (C, D) ∆Gelec hypersurfaces in the Na2Asp-bound and apo states. The y-axis represents the insertion level of the transporter in the membrane dielectric. (E) Maximum barrier height for transition between several transporter states. (F) Computed valence along the reaction coordinate. ∆Gelec was calculated by solving the linearized Poisson-Boltzmann equation using the APBS routine. 175x307mm (300 x 300 DPI)



Figure 7: Reshaping of the electric field during the outward- to inward-facing transition. Electrostatic calculations of the iso-potential lines (A, C, E) were performed using APBSmem with an internal potential 100 mV and an external potential of 0 mV, after setting all protein charges to 0. Structures were obtained through steered MD (B, D, F). The intermediate structure represents the translocation progress at the energy minimum, Fig. 6B, at about 35% completion of translocation. 176x205mm (300 x 300 DPI)


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Figure 8: A minimal transport model with charge compensation and extracellular bowl can explain the data. (A) Illustration of the model with extracellular bowl with negative charges shown in red and positive charges shown in blue. The wire mesh indicates the transition from ε = 2 for the membrane and ε = 80 for the solvent. (B) Polar (electrostatic) solvation energy as a function of the reaction coordinate.(C, D) ∆Gelec hypersurfaces in the fully loaded state in the presence and absence of the bowl. The y-axis represents the insertion level of the transporter in the membrane dielectric. 124x161mm (300 x 300 DPI)



Figure 9: Electrostatic energy contribution to translocation in EAAT3. ∆Gelec hypersurfaces in the Na3Aspbound state. The y-axis represents the insertion level of the transporter in the membrane dielectric. The transition was modeled using a EAAT3 homology model based on the GltPh structure and steered MD, applying force to the Cα atoms of the substrate and R446. 271x207mm (300 x 300 DPI)


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TOC graphic 270x353mm (300 x 300 DPI)


Energy Landscape of the Substrate Translocation Equilibrium of

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