The Environment Shapes the Inner Vestibule of LeuT

Abstract

Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of ~15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure.

Fig 1. Comparison of residue mobility (β-factor).

Comparison of β-factors extracted from crystal structures (outward-occluded: 2A65; inward-open: 3TT3) with β-factors calculated from MD simulations for (A) the membrane inserted outward-occluded LeuT, (B) membrane inserted inward-open LeuT and (C) micelle inserted inward-open LeuT. (D-F) Zoom into the region of TM1A (highlighted in yellow). The color code of black, green, purple and cyan is used throughout the manuscript to identify respectively run 1, 2, 3 and the crystal structure. (G) The structure of the inward-open LeuT is color coded (from red to blue) by β-factors as reported in the crystal structure (PDB ID: 3TT3).

Fig 2. Dynamics of helix TM1A.

(A) Comparison of the final structures of three independent simulations (grey, green, purple) of membrane-inserted LeuT with the inward-open crystal structure (PDB ID: 3TT3) shown in cyan. Lipid molecules are shown in grey, the dark spheres represent the phosphate atoms of the membrane lipids. (B) Change in vestibule size of membrane inserted LeuT over time is quantified by measuring the distance between the Cα of residue M18 (TM1A) and Y265 (TM6). The values of each time frame (thin line) are shown together with a running average over 100 frames (thick line). The distances as observed in the crystal structure of LeuT (dashed line for the inward-open structure with the PDB ID: 3TT3; a dotted line for the outward-occluded structure with the PDB ID: 2A65) are also shown. (C) Comparison of the final structures of three independent simulations (grey, green, purple) of micelle-inserted LeuT with the crystal structure (PDB ID: 3TT3) shown in cyan. Detergent BOG molecules are shown in yellow, atom O1 as orange spheres. (D) Change in vestibule size as described in B for detergent solubilized LeuT. (E, F) The size of the vestibule is shown for the final conformation of run 2 of membrane-inserted LeuT (E) and of run 2 of micelle-inserted LeuT (F). The protein is shown in green, the pore surface in blue, as calculated by the program caver 3.0.

Fig 3. Water density in LeuT.

Water (blue) and phosphate (of POPC lipids) density (brown) were averaged over the 200 ns of run 1. The starting structure of LeuT is shown, the substrate leucine (cyan), the salt bridge of the outer vestibule (magenta), R11 (pink) and the O1 atoms of BOG (orange) are highlighted. (A) Water penetrates until the salt bridge of the extracellular gate (R30 and D404) in the outward-occluded state. (B) Opening of the inner vestibule allowed water to reach the substrate of the inward-open LeuT. (C) Similar water penetration was observed in the micelle embedded inward-open LeuT. (D) The physico-chemical properties of all residues are mapped onto the structure of the inward-facing structure using a color code: non-polar (white), polar (green), positively charged (blue), negatively charged (red).

Fig 4. Movement of sodium 1 and substrate of the inward-open conformation.

Panels A, B show the displacement of sodium 1 and substrate from three independent simulations of the membrane inserted inward-open LeuT. Panels C, D show the respective movement of the micelle embedded LeuT. Panels E-H: The final structures of three independent simulations of 200 ns duration each are shown along with the starting structure for the membrane inserted LeuT in cyan (E, G) and micelle embedded (F, H). Please note, the final positions of Na1 are essentially identical in two (green and gray) simulations in panel E.

Fig 5. LRET based distance measurements.

(A) Schematic rendering of the LRET experiment. The Tb⁺³donor is in complex with the LBT tag (red) inserted at the C-terminus; the BODIPY-C3-M acceptor dye (magenta) is chemically linked to residue 9 at the N-cap motif TM1A. (B) Representative Tb⁺³ decay traces either incorporated into micelles or reconstituted into proteoliposomes, either unlabeled or labeled with the BODIPY-C3-M acceptor dye, in the presence of 200 mM Na⁺ or Na⁺ free. The structure of BODIPY-C3-M is shown in the insert. (C) Distances calculated from the donor decay between the Tb⁺³ and the acceptor dye. Shown are means ± S.E.M. from three independent experiments done in triplicate.

Fig 6. Potential of Mean Force (PMF) of TM1A movement.

The center of mass distance between the Cα atoms of TM1A (residues 11 to 20) and the Cα atoms of IL1 (residues 78–81), TM8 (residues 363–366) and TM12 (residues 503–504) was used as reaction coordinate. The PMFs were constructed using umbrella sampling combined with the weighted histogram analysis method, error bars were estimated by applying the bootstrap method. For simplicity, only every ~15^th error bar from the bootstrap results is shown. The distance measured in the inward-open crystal structure is shown as vertical cyan line.

Fig 7. Synthesis scheme for BODIPY-C3-M.

a) MeOH/DCM (2:1), LiOH (aq), rt, 4h; b) DCM, tosyl chloride, NEt₃, DMAP 0°C, rt; c) Phthtalimide potassium salt, MeCN (5% DMSO), 50°C, 24 h; d) 1. Hydrazine, EtOH/DCM; 2. DCM, N-methoxycarbonylmaleimide, rt, 52 h.