Electrostatic fields.

<p>A cytosolic view and a side view is shown for a representative hDAT dimer structure for each cluster, indicated by the labels A-H. The hDAT surfaces are shown in white, while fields generated are shown in blue (positive potential) and red (negative potential). Potential surfaces are drawn at 2eV.</p

Dopamine transporter oligomerization involves the scaffold domain, but spares the bundle domain

<div><p>The human dopamine transporter (hDAT) is located on presynaptic neurons, where it plays an essential role in limiting dopaminergic signaling by temporarily curtailing high neurotransmitter concentration through rapid re-uptake. Transport by hDAT is energized by transmembrane ionic gradients. Dysfunction of this transporter leads to disease states, such as Parkinson’s disease, bipolar disorder or depression. It has been shown that hDAT and other members of the monoamine transporter family exist in oligomeric forms at the plasma membrane. Several residues are known to be involved in oligomerization, but interaction interfaces, oligomer orientation and the quarternary arrangement in the plasma membrane remain poorly understood. Here we examine oligomeric forms of hDAT using a direct approach, by following dimerization of two randomly-oriented hDAT transporters in 512 independent simulations, each being 2 μs in length. We employed the DAFT (docking assay for transmembrane components) approach, which is an unbiased molecular dynamics simulation method to identify oligomers, their conformations and populations. The overall ensemble of a total of >1 ms simulation time revealed a limited number of symmetric and asymmetric dimers. The identified dimer interfaces include all residues known to be involved in dimerization. Importantly, we find that the surface of the bundle domain is largely excluded from engaging in dimeric interfaces. Such an interaction would typically lead to inhibition by stabilization of one conformation, while substrate transport relies on a large scale rotation between the inward-facing and the outward-facing state.</p></div

Representative dimer structures.

<p>A side view of a representative dimer structure is shown for each cluster (A-H). The insert zooms into the interface region from the same membrane side view or from the membrane plane. Residue side chains in contact with the second protomer are highlighted.</p

Potential of Mean Force.

<p>Panels A-H show PMF profiles of protomer separation of two dimers per cluster, revealing the stabilization energy of each dimer. Panel I shows the essentially flat PMF profiles for every transient dimer that dominantly interacted with the bundle domain during the DAFT simulations (See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006229#pcbi.1006229.g003" target="_blank">Fig 3B</a>). A PMF profile for the respective system was calculated starting from the frame of the DAFT simulations with the smallest hDAT-hDAT distance. The same color code was applied as used in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006229#pcbi.1006229.g003" target="_blank">Fig 3B</a>.</p

Schematic representations.

<p>Representative system showing randomly oriented A) starting (0 μs) and B) end (2 μs) structure in coarse-grained representation. For the protein, only the backbone of the transmembrane helices and the C-terminal helix are shown. The transporter is visualized from the intracellular side. C) Schematic representation to illustrate the quantification of relative hDAT dimer orientation. The transporter is visualized from the intracellular side. The red arrow represents the reference vector or direction within the frame of individual hDAT transporter. It is oriented roughly towards TMH4 and located within the plane of the membrane. The angle β (cyan area) quantifies the center of mass position of protomer B relative to the reference frame in protomer A by measuring the angle between the reference vector of Protomer A and the line connecting the center of masses of both protomers. The angle χ (yellow area) determines the center of mass position of protomer A relative to the reference frame in protomer B by measuring the angle between the reference vector of Protomer B and the line connecting the center of masses of both protomers.</p

Conformational changes during transport.

<p>The schematic representation shows hDAT A) in the outward-open and B) in the inward-open conformation, exemplifying the changes in protein geometry during transport. C) Visualization from the intracellular side of the scaffold (cyan) and bundle domains (magenta) on a surface representation of the outward-facing conformation. The orientation in panel C is identical to the orientation of reference protomer in Figs <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006229#pcbi.1006229.g003" target="_blank">3</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006229#pcbi.1006229.g004" target="_blank">4</a>.</p

Synthesis scheme for BODIPY-C3-M.

<p>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, <i>N</i>-methoxycarbonylmaleimide, rt, 52 h.</p

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

<p>Panels <b>A, B</b> show the displacement of sodium 1 and substrate from three independent simulations of the membrane inserted inward-open LeuT. Panels <b>C, D</b> show the respective movement of the micelle embedded LeuT. <b>Panels E-H</b>: 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 (<b>E, G</b>) and micelle embedded (<b>F, H</b>). Please note, the final positions of Na1 are essentially identical in two (green and gray) simulations in panel E.</p

LRET based distance measurements.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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.</p

Dynamics of helix TM1A.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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. <b>(D)</b> Change in vestibule size as described in B for detergent solubilized LeuT. <b>(E, F)</b> The size of the vestibule is shown for the final conformation of run 2 of membrane-inserted LeuT <b>(E)</b> and of run 2 of micelle-inserted LeuT <b>(F)</b>. The protein is shown in green, the pore surface in blue, as calculated by the program caver 3.0.</p