Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer

Abstract

Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.

Keywords: conformational dynamics; hydrogen–deuterium exchange mass spectrometry; molecular dynamics simulations; nanodisc; neurotransmitter symporter.

Fig. 1.

Overall structural comparison and peptide coverage. (A) Outward-open LeuT structure (PDB ID code 3TT1; gray) superimposed onto inward-open LeuT (PDB ID code 3TT3; colored) using TMs 3, 4, 8, and 9 [the so-called “hash motif” (83)]. Color correlates to positional change (0–19 Å) in the Cα atoms of aligned residues. (B) Inward-open LeuT structure colored to represent the peptides monitored in this study. Color reflects redundancy of the amino acid in the final peptide list.

Fig. S1.

Schematic of the conformational cycle of LeuT (green). Substrate, sodium ions, and proton are depicted as a blue hexagon, red circles, and a black circle, respectively.

Fig. S2.

Saturation binding of [³H]Leu to nanodisc-reconstituted LeuT-WT (green circles), -Y268A (orange), and empty nanodiscs (blue triangles). Data were fit to a rectangular hyperbola, as implemented in GraphPad Prism 7.01. Error bars represent the SEM for each data point over three independent experiments.

Fig. S3.

LeuT peptides observed during sequence identification runs mapped onto the amino acid sequence of the protein. Peptides are represented as blue bars and aligned to their relative position along the primary sequence.

Fig. 2.

Deuterium uptake kinetics of representative peptides from LeuT. Plots depict data from the outward-favoring (green) and inward-favoring (orange) conditions for peptides across LeuT. The structure (center) is colored to highlight the locations of the peptides in TM1a (blue), EL2 (purple), TM7 (brown), EL4a (yellow), the EL4ab loop (orange), and EL4b (red).

Fig. 3.

Difference in deuteration profiles of LeuT peptides between outward- and inward-favoring conditions. Bars represent the difference in total deuteration [over four time points, represented as lines: 10 s (cyan), 60 s (blue), 600 s (purple), and 7,200 s (black)], with negative values (orange) indicative of higher deuteration in the inward-favoring condition. Gray regions indicate 98% confidence interval cutoffs (Materials and Methods) for individual time points (dark gray) and the total (light gray). NT, N terminus; CT, C terminus.

Fig. 4.

EX1 kinetics of EL2 and TM7. (A) Superimposed structures of outward- and inward-open conformations of LeuT highlighting the locations of EL2 and TM7. (B and C) Mass over charge spectra (black lines) measured without deuteration (UD) and after deuteration for four different time windows, for peptides 142–148 (EL2; B) and 278–285 (TM7; C) arranged vertically to depict the difference in progressive deuteration in the outward- (WT; Left) and inward-favoring (Y268A; Right) conditions, respectively. The total isotopic peak (blue) was decomposed into protected (green) and unfolded (red) fractions, when applicable.

Fig. S4.

Calculated protection factors for the MD simulations of LeuT. (A) Convergence of the total protection factor over all residues in each simulation (Materials and Methods) was computed as a function of the input simulation time window, for each of three trajectories (red, green, blue circles), and as a mean and SD of the three (black circles) for either outward-open (Upper) or inward-open (Lower) conformations. For reference, the corresponding total protection factors for the input X-ray structures are 60.9 × 10⁸ (3TT1; outward-open) and 7.6 × 10⁸ (3TT3; inward-open); the higher degree of protection presumably reflects the lack of thermal fluctuations and dehydration of the crystal. (B) Location of the least (red) and most (blue) protected regions in simulations of the outward-open (Left) or inward-open (Right) conformations of LeuT. The protection factor for individual residues was computed using all three trajectories for their full lengths, and the log of the protection factor was mapped onto the input structures.

Fig. 5.

Agreement of deuterium uptake trends over 21 LeuT peptides between in vitro (green and orange) and in silico (blue) HDX experiments. Left and right panels depict data for deuterium uptake for peptides in the outward- and inward-favoring states of the transporter, respectively, compared with simulation data starting with the outward- and inward-open states, respectively.

Fig. S5.

Comparison of deuterium uptake between in vitro (Exp) and in silico (Sim) experiments represented as heat maps on crystal structures of LeuT in the outward-favoring (A) and inward-favoring (B) conformations for the undeuterated (0 s) system and after four different deuterium exposure time points.

Fig. 6.

Behavior of TM1a in simulations of inward- and outward-facing LeuT. (A) Outward-open LeuT structure (PDB ID code 3TT1; green) superimposed onto the inward-open structure (PDB ID code 3TT3; red-orange) highlighting TM1a as opaque cartoon helices. (B) Angle of TM1a relative to the membrane normal (z axis) during MD simulations of the two structures, shown as the time dependence for each of three trajectories of the outward- (black, red, cyan lines), and inward-facing conformations (green, orange, blue lines), sampled every 100 ps, and compared with the values computed for the X-ray structures (red diamonds at t = 0). Data sampled every 10 ps are shown in gray. For each time frame, the protein was superposed onto the initial structure using the Cα atoms of TM helices 3, 4, 8, and 9 (the hash domain); the starting structure, in turn, was aligned to the approximate membrane plane according to OPM (Materials and Methods). (C and D). Snapshots of LeuT in outward- (green; C) and inward-open (orange; D) states, embedded in a hydrated DMPC bilayer (spheres). Protein molecules are depicted as ribbons and surface representation, with the surrounding water molecules shown as blue sticks.

Fig. S6.

Aqueous accessibility of residues in the cytoplasmic pathway of LeuT during simulations of the outward- and inward-open structures. (A) Simulation snapshots from the end of the trajectories, viewed from along the membrane plane and with the periplasmic side toward the Top. The water volume computed as the occupancy over the last 50 ns of three trajectories is shown as purple density at 30% occupancy level. The protein is shown as cartoon helices, with TM1a (blue) and TM7 (brown) highlighted. Helices in the scaffold (TMs 3, 4, 8–12) are omitted for clarity. Amino acids whose solvent accessibility increases in the simulations of the inward-open structure are shown as sticks, except for nonpolar hydrogen atoms. (B) Relative contribution of amino acids at the N terminus and in TM1 toward the increased deuteration observed in the inward-favoring condition. Graph shows difference in #D between the two conditions for three overlapping peptides [residues 1–12 (orange), 1–14 (brick), and 1–16 (red)] over four time points. Inset shows deuteration kinetics for peptide 18–28 (purple bar) for the outward-open (green line) and inward-open (orange line) states, respectively. (C) Surface area of the backbone (Top) or side chain (Bottom) of each residue that is accessible to water during simulations of the outward-open (green) and inward-open (orange) states, computed as the mean and SD over all snapshots taken every 100 ps from all three trajectories. Residues of TM1 (blue) and TM7 (brown) that are helical in the outward-facing structure (3TT1) are highlighted. Residues in TM1 and TM7 whose solvent-accessible surface area (SASA) increases significantly in the inward-open state are indicated with arrows.

Fig. 7.

Altered dynamics of EL4 in LeuT under outward- and inward-favoring conditions. (A) Mass over charge spectra measured without deuteration (UD) and after deuteration for four different time windows, for a peptide in EL4 comprising residues 312–324, indicating a unique population of more highly deuterated peptides in inward-favoring (Right) relative to outward-favoring LeuT (Left). The total isotopic peak (blue) is decomposed into protected (green) and unfolded (red) fractions, when applicable. (B) Position of EL4a and EL4b in the outward-open (orange) and inward-open (green) structures of LeuT. The position of A319 and its neighbor G318 (ball-and-sticks) are shown in detail in the Inset. (C) Fraction of peptide 312–324 deuterated (unfolded) as a function of incubation time for LeuT-WT (orange) and -Y268A (green) LeuT. (D) Backbone (θ, Upper, and ψ, Lower) dihedral angles in A319 in the EL4ab loop during MD simulations of the outward-open (3TT1; Left) and inward-open structures (3TT3; Right). For each simulation system, three independent trajectories are shown (black, red, and cyan points) sampled every 100 ps. For reference, the θ, ψ values of the X-ray structures are as follows: –58.3°, –46.7° in 3TT1; and –84.0°, 141.2° in 3TT3.

Fig. S7.

Location (blue) of peptides whose spectra exhibited a static double-envelope behavior in either outward-favoring (green) or inward-favoring (red) condition, are mapped onto the X-ray structures of outward- and inward-facing conformations, respectively.