Dopamine transporter oligomerization involves the scaffold domain, but spares the bundle domain
[['The human dopamine transporter ( hDAT ) is a member of the monoamine transporter family [1] , which also includes the transporters for serotonin ( hSERT ) and noradrenaline ( hNET ) .', 'Within the central nervous system , these transporters are localized to pre-synaptic neurons close to the synaptic junctions , but primarily outside the synapse [2] .', 'Their role is to efficiently curtail neurotransmitter-mediated signaling via uptake of associated neurotransmitters into pre-synaptic neurons .', 'These transporters utilize the sodium/chloride gradient across the plasma membrane to provide the driving force for transport [3] .', 'Dysfunction in hDAT leads to severe neuronal disorders , such as Parkinson’s disease , attention-deficit hyperactivity disorder ( ADHD ) , depression and schizophrenia [4 , 5] The first crystal structure for this class of transporters became available for the bacterial homolog small amino acid transporter ( LeuT ) isolated from Aquifex aeolicus [6] .', 'The Drosophila melanogaster dopamine transporter ( dDAT ) was the first eukaryotic member of the SLC6 family to be resolved by X-ray crystallography [7] , followed more recently by crystallization of the human serotonin transporter ( hSERT ) [8] .', 'LeuT was crystallized in three conformations [6 , 9 , 10] , revealing that during the transport cycle the bundle domain ( consisting of transmembrane helices TMH 1 , 2 , 6 , 7 ) rotates by ~30° relative to the scaffold domain ( TMH 3 , 4 , 8 , 9 . 10 , 12 ) , thus anchoring the transporter to the membrane .', 'TMH 5 and 11 are also assigned to the scaffold domain , though they bend during the conformational transition of the transport cycle .', 'The similarity of these crystal structures confirmed that several findings can be conveyed from the bacterial LeuT to the human homologs , including transporter topology , conformational changes of the transport cycle [6 , 9–11] and the substrate binding site [12] .', 'These structures also revealed important differences , as the human transporters do not share the dimeric form with LeuT , due to the conformation of transmembrane helix 12 ( TMH12 ) [6 , 7] .', 'TMH12 is central in the dimer interface of LeuT , but is remarkably different in dDAT and hSERT .', 'Cross-linking experiments and Förster resonance energy transfer ( FRET ) measurements indicated the existence of higher-order oligomers of human monoamine transporters [13–20] in the plasma membrane .', 'Functional studies revealed the importance of higher-order oligomers for their physiological function , and also revealed substrate-transport and inhibitor binding-dependent oligomerization [19 , 21–25] .', 'Spectroscopic studies of hSERT showed that the transporter assumes a large distribution of oligomeric sizes [26 , 27] .', 'The stability of the oligomer and protomer exchange rates were contingent on the presence of phosphatidylinositol-4 , 5-bisphosphate ( PIP2 ) [28] .', 'Monoamine transporters have mainly been simulated in their monomeric form , although LeuT-based dimers have also been examined [29 , 30] .', 'Recently , protein docking predicted a single hDAT dimer conformation that included residue C306 in the interface , which was assessed using MD simulations [30] and site-directed mutagenesis studies .', 'In the current study , our key aim was to develop a comprehensive description of hDAT oligomerization by computational approaches .', 'We hence characterized the interfaces of human DAT ( hDAT ) oligomers and identified the transmembrane helices and residues involved using a hDAT model based on the outward-facing dDAT crystal structure .', 'Membrane properties , including lipid entropy , play a crucial role in the process of oligomerization .', 'We have therefore used the unbiased DAFT ( Docking assay for transmembrane components ) [31] approach to directly follow the formation of hDAT dimers , starting from two randomly-oriented membrane inserted hDAT transporters .', 'We observed the formation of four stable symmetric and four asymmetric dimer conformations .', 'Importantly , we found only a very modest contribution of the bundle domain in the dimer interfaces , consistent with its role as a moving domain during the transport cycle .'], ['Relative orientation of the two protomers in the dimer was assessed through the introduction of an internal coordinate system , as shown in Fig 1C .', 'We first defined an orientation vector as the reference vector ( red arrow ) within the frame of each hDAT molecule .', 'The position ( β ) of protomer B relative to protomer A is measured as the angle between the reference vector and the vector connecting the center of mass of the two hDAT monomers .', 'In addition to the orientation described in the β angle , the second hDAT ( protomer B ) molecule can also rotate around it own axis .', 'Therefore , we introduced a second angle ( χ ) that measures the orientation of the reference vector of protomer B relative to the line connecting the center of mass of the two hDAT molecules .', 'The orientation plot ( Fig 2 ) summarizes across the ensemble the relative orientation of the two protomers using these two angles ( β and χ ) .', 'The orientation plot therefore reflects the frequency with which each orientation was observed , while not averaging over the symmetry-related orientation of the homodimers .', 'The series of plots in Fig 2 shows the time evolution of orientations at 0 . 0 μs , 0 . 5 μs , 1 . 0 μs , 1 . 5 μs and 2 . 0 μs .', 'Over time an enrichment of a few prominent clusters or orientations became apparent , visible as red-to-yellow regions , while the frequency of observing other conformations decreased , which are indicated by blue colored areas , in which the dimer probability is below average .', 'The orientation plot is symmetric with respect to the diagonal once converged , because we measure the orientation of homodimers .', 'Clusters on the diagonal represent symmetric dimers , while all off-diagonal dimers are asymmetric , differing in the helices in the shared interfaces .', 'We did not impose symmetry in the analysis , but rather used it as a measure of convergence .', 'The inherent symmetry of the orientation plot requires that at full convergence all off-diagonal peaks must be present in both symmetry-related positions and must be sampled with the same frequency .', 'The plot at 2 . 0 μs was remarkably symmetric , indicative of an almost fully converged dataset in which the appearance of additional conformations at even longer simulation times is unlikely .', 'The time evolution of the degree of symmetry of the four off-diagonal clusters E-H showed that the ratio converges from 1:3 at 0 . 5 μs to 1:1 . 2 at 2 . 0 μs .', 'The ratios were estimated by calculating the difference between the number of systems in the four clusters above the diagonal over the number of systems in the four clusters below the diagonal .', 'The second applied measure of convergence was the time evolution of the potential energy of hDAT-hDAT interactions ( S1 Fig ) .', 'The plot showed that interacting dimers were present in large numbers and the profile is leveling off .', 'Exchange between dimer conformations with the same interaction energy cannot be detected by the interaction energy plot , but would remain visible in the orientation plot .', 'The plateau in the interaction energy plot is reached once the number of interacting dimers in the full ensemble has equilibrated and the populations of weaker and stronger interacting dimers no longer change .', 'Experimental data for the parolog human SERT have shown that ~35% of all hSERT transporter are monomeric in the endoplasmatic reticulum ( ER ) membrane as well as in the plasma membrane [27 , 28] .', 'The ER membrane is similar to our bilayer in that it mainly consists of phosphatidyl lipids , while being almost devoid of cholesterol and free of PIP2 .', 'Given the similarity between hSERT and hDAT we should also not expect that all hDAT transporters in the ensemble interact and form dimers .', 'When comparing frequencies between symmetric and asymmetric dimers , the two symmetry-related off-diagonal conformations of an asymmetric cluster must be added together , while for the diagonal clusters these are already integrated , because of overlapping on the diagonal in the plot .', 'In total , we found four symmetry-related conformations on the diagonal ( cluster A-D ) and four asymmetric conformations not on the diagonal ( cluster E-H ) were observed ( See S1 Table for number cluster members ) .', 'The orientation plot showed that hDAT dimers clustered into a limited number of conformations .', 'To set these into a structural perspective , we fitted all final structures ( 512 structures ) to the first protomer and analyzed the position of the second protomer .', 'The distribution of the second protomer was , as expected , not uniform throughout the ensemble ( S2 Fig ) .', 'In Fig 3A we show an overlay using one representative dimer per cluster ( Fig 2D ) .', 'All dimers were fitted to protomer A to place all 8 clusters into a single reference frame .', 'Position and orientation of protomer B therefore shows its relative arrangement in the hDAT dimers .', 'Importantly , this overlay revealed that possible dimer interfaces included a large part of the membrane-exposed hDAT surface , enclosing the transmembrane helices of the scaffold domain , but also highlighted a prominent exception , which was adjacent to the bundle domain .', 'A comparison with the orientation plot ( Fig 2 ) revealed that the frequency of finding dimers that included the bundle domain within the interface ( corresponding to the region of ~105–150 ° ) was decreasing over time .', 'We then screened all trajectories to investigate if any dimer was formed in which the bundle domain was central for the dimer interface at any time-point throughout the 2 μs long simulations .', 'In a condition of complete random encounters , we should have expected 20–40 contacts , but we found only 7 such trajectories ( 1 . 4% of all simulations ) .', 'In all seven simulations , these dimers were unstable and separated within 0 . 5 μs ( Fig 3B ) .', 'The orientation plot showed the formation of 8 distinct clusters .', 'One representative dimer was extracted from every cluster , converted into fine-grained representation and simulated for 100 ns to test their stability at the all atom resolution of classical force fields .', 'These systems proved to be stable , confirming that the structure obtained through the DAFT workflow at the coarse-grained representation are stable low-energy conformations ( S3 Fig ) .', 'Fig 4A and 4B show representative structures for every cluster , whereby all dimers are fitted to protomer A . The position of protomer B is thereby shown ( and indicated in degrees ) using the internal coordinate system as references , as is also used in Fig 2 .', 'Comparison between Fig 4A and 4B shows that the binding geometry of some clusters like cluster C and G have mutually exclusive conformations , while clusters A and G could in principle contemporaneously form hDAT-hDAT dimers , leading to higher oligomeric structures .', 'Analysis of the transmembrane helices and loops within the interfaces is summarized in Fig 4C .', 'The axes of Fig 4C are labeled by residue numbers; transmembrane helices and ( frequently observed ) loops are indicated next to the axes , colored according to the colors used for all DAT structures .', 'Fig 4C shows a contact heat map at single residue resolution , integrated over the last 100 ns of each trajectory .', 'Contact frequencies between residues are shown in the triangle above the diagonal coded in gray scale .', 'It shows that some interactions are very frequent , indicating that these interactions could be essential for dimer stability .', 'We find that transmembrane helices TMH1 , 2 , and 7 are completely absent in any interface .', 'TMH6 can be found in the interface , although it is limited to interactions of residues of the first helical turn .', 'The lower panels color residue contacts according to association to one of the clusters .', 'Overall , most contact on or close to the diagonal are from symmetric dimers .', 'A per residue analysis ( S4 Fig ) shows interactions and interaction frequency for all dimers associated with one of the clusters .', 'The symmetric dimers include less transmembrane helices within the interface regions than the asymmetric dimers .', 'This was expected , because in the symmetric assembly the same regions are present on both protomers .', 'On average , the interfaces of the symmetric dimers consisted of 1–2 transmembrane helices or loops on each protomer surface .', 'We can infer from experimental data on the hSERT [28] that showed exchange rates of protomers on a time scale of minutes , that dimer association and dissociation are slow processes .', 'Interestingly , the exchange rate was comparable between the cholesterol-free ER membrane and the cholesterol-containing plasma membrane , if devoid of PIP2 , indicating that the exchange rate is not very sensitive to the membrane composition .', 'Sequential residues along a helix change orientation by ~100° relative to the main helix axis .', 'The interaction of helices therefore follows an alternating pattern , which is visible in Fig 4C as a strand that is oriented parallel to the diagonal ( for parallel-oriented helices ) or normal to the diagonal ( for antiparallel-oriented helices ) , while the interacting residues show the typical heptad repeat pattern ( S4 Fig ) .', 'The length for the pattern is representative for the extent of helix-helix interactions .', 'The symmetric interfaces of clusters B and D were dominated by interactions of residues outside transmembrane helices ( Fig 5 ) .', 'Cluster B was mainly stabilized by interactions between the EL2 loops at the extracellular site , whereby the highly-conserved residues W184 and N185 were found in the core of the interface .', 'It was shown experimentally that a mutation of W184 leads to a loss of surface expression [33] .', 'Possible explanations might be a folding defect , which would lead to transporter degradation or a lack of transporter dimerization , which is required for ER export .', 'Several lipids filled the space between the protomers .', 'A similar lipid-bridged dimer was observed for cluster D , in which the protomer interactions were dominated by interaction between the C-terminal helices at the intracellular site .', 'The dimer interface was stabilized by the salt bridge between R588 and E589 across the dimer interface , and further stabilized by additional interactions between aromatic residues on the C-terminal helix .', 'The configurations of clusters B and D shared an additional property .', 'The EL2 loop and the C-terminal helix were immersed in the membrane , and thus strongly interacted with the lipid headgroup region .', 'In addition to direct interactions , the immersion perturbed the membrane bilayer , which could contribute to the attractive dimer-stabilizing forces due to membrane deformation .', 'In contrast , the symmetric interfaces of clusters A and C established transmembrane contacts ( Fig 5 ) .', 'In cluster A , these comprised the entire TMH9 , while TMH4 contributed mainly at its extracellular side .', 'The geometry of the interface in cluster C was less specific , as TMH6 and TMH11 were within the interface in only a subset of the dimers .', 'Common to both clusters was the fact that they included the cysteine residues C243 and C306 , which have been shown by cross-linking experiments to be part of interfaces [13 , 14] .', 'Cluster C showed dimers , which were stabilized by symmetric inter-molecular salt bridges between R304 and E307 .', 'These interactions oriented the two C306 residues to face each other at cross-linking distance .', 'The conformation in cluster C was mainly stabilized by this pair of salt bridges , as the shared surface is otherwise small , leaving space to allow for dimer plasticity , mobility and dynamics .', 'The off-diagonal clusters in the orientation plot are asymmetric and therefore share different interfaces ( Fig 5 ) .', 'The orientation plot ( Fig 2 ) showed four main off-diagonal clusters and also revealed that the interface on one protomer was able to interact with more than one interface on the second protomer .', 'Cluster E1/2 has several properties in common with cluster B , including the EL2 loop that is prominently present in the interface and the lack of extensive contacts in the hydrophobic core of the membrane .', 'The EL2 loop interacted with TMH3 and 4 on the first protomer and TMH5 on the second .', 'Cluster H1/2 shared the EL2 loop as part of the interface , but the orientation of the second protomer was remarkably different from cluster E2/3 , as the contacting interface was mainly TMH12 , though limited to its extracellular side .', 'The A559V mutation is located within the extracellular region of TMH12 and is associated with Autism spectrum disorders ( ASD ) [34 , 35] , caused by trafficking and functional alterations , while the P554L mutant , located in EL5 , is associated with the Dopamine Transporter Deficiency Syndrome ( DTDS ) [36] and has been described as intracellularly retained .', 'The dimer clusters ( F1/2 and G1/2 ) formed an extensive interface throughout the transmembrane region .', 'Both clusters shared TMH11 in their interface , often including contacts to EL3 .', 'TMH11 has been reported to be located in dimerization interfaces [17] of human monoamine transporters .', 'The main helix interfacing with TMH11 was TMH5 in cluster F1/2 and TMH4 in cluster G1/2 .', 'This change in relative orientation is achieve by a ~60° rotation of the second protomer .', 'Cluster G1/2 is of particular interest , because residue C243 and C306 were found at cross-linking distance from each other .', 'Cross-linking experiments showed that hDAT can form asymmetric trimer and tetramers [14] through a chemical bond established between C243 and C306 .', 'We quantified the stability of observed dimers by determining Potential of Mean Force ( PMF ) profiles for dimer dissociation of two representative dimers per cluster ( Fig 6 ) by applying the same CG system representation as used for the DAFT simulations .', 'The starting structures of all systems are shown in S5 and S6 Figs .', 'In the first step , the two protomers were pulled apart by steered MD ( SMD ) simulations in a 100 ns long trajectory , while restraining the relative orientation using the enforced rotation module .', 'The applied pulling velocity ( 0 . 025 nm/ns ) was set to be in the range of the fastest diffusion observed in the unbiased simulations .', 'The rationale for selecting such a slow pulling rate was to use a velocity that is compatible with normal diffusion in the membrane in order to obtain SMD trajectories that minimally perturb the membrane .', 'In the second step we extracted equidistant structures at 0 . 1 nm separation over 1 . 6 nm and carried out PMF calculations using umbrella sampling .', 'Additional conformations with 0 . 025 nm increments over the first 0 . 4 nm were added in the initial rising part of the PMF to improve sampling and achieve sufficient histogram overlaps .', 'Overall , the PMF profiles of dimers extracted from all clusters showed qualitatively similar profiles , with a dimer-stabilizing potential between 30 and 90 kJ·mol-1 ( Fig 6 ) .', 'S2 Table reports the integrals of each PMF profile .', 'The error bars reflect the variability observed in the PMF calculations and represent lower boundaries , because additional variability which might only appear over a much longer timescale is not sampled in the umbrella window calculations .', 'The restraining forces of the enforced rotation module applied to prevent translation ( S7 Fig ) and rotation ( S8 Fig ) , showed a Gaussian-like distribution , which indicates that the restraints did not mask any hidden energy gradient or forces .', 'To provide a better estimate of the variability of the PMF profiles , we calculated two PMF profiles per cluster , using two different hDAT dimers as starting structures .', 'The profiles of clusters D-H showed limited difference between the two profiles , suggesting that these are representative profiles .', 'The profiles for the clusters A , B and C showed a large difference between the two profiles .', 'The very large energy difference between the dimeric and the monomeric state of one profile of cluster A indicates that this profile might be overly attractive .', 'The difference between the two profiles of cluster B and C are more likely a consequence of differences in the starting structures , which were selected according to the centrality of each cluster , but differed in the details of their interfaces ( S5 and S6 Figs ) .', 'The dimers of clusters B , D , but also F , showed weaker and broader interaction profiles of 20–50 kJ·mol-1 .', 'The dimers with a large transmembrane interface exhibited especially strong and short-range interaction profiles , showing 60–75 kJ·mol-1 of stabilizing energy .', 'The short-range nature of these profiles indicate that they are dominated by van der Waals interaction between transmembrane helix side chains , while the broader profile indicates an important role of lipids and/or the changes on the intracellular and extracellular loops .', 'Of particular importance were the details of loop interactions across the dimer interface as obtained from the final structure of the DAFT simulations .', 'The broader minima were associated with the sliding of residues from the entangled opposing loops during the initial phase of separation .', 'The PMF profiles were strikingly different for the 7 dimers that formed transient interactions involving the bundle domain ( Figs 3B and 6I ) .', 'From every unbiased trajectory we extracted the frame with the smallest dimer-dimer distance as a starting point for the PMF calculations .', 'All seven profiles showed a flat energy landscape , revealing that these structures were not held together by any stabilizing interactions , therefore transient and short-lived .', 'Binding of the negatively-charged lipid PIP2 was shown for hSERT to affect oligomerization [28 , 37 , 38] .', 'The protomers in each oligomer exchanged with a timescale of minutes in both the plasma membrane ( if depleted of PIP2 ) and the ER .', 'The presence of PIP2 in the plasma membrane blocks this exchange process and kinetically traps the oligomer in its state at the plasma membrane .', 'PIP2 carries a charge of -5 .', 'We thus assumed that binding is dominated by electrostatic interactions and calculated the electrostatic potential for each cluster ( Fig 7 ) using apbs [39] .', 'A representative structure for each cluster was converted from the coarse-grained representation to a fine-grained all-atom structure using the back-mapping module [40] by applying two steps of energy minimization and four steps of system relaxation .', 'Visualization of the potential isosurfaces at 2 eV revealed potential PIP2 binding sites as areas of a positive electrostatic potential extending into the membrane .', 'A large positive electrostatic field reaching into the membrane was generated by three clusters: the symmetric cluster C , which includes C306 in its interface at the extracellular site , the asymmetric clusters F1/2 and G1/2 , which both showed extensive contacts across the lipid bilayer .', 'The respective fields were generated by the positively-charged lysine and arginine residues of IL5 for the symmetric cluster C , by residues in IL3 , IL5 and the N-terminus for cluster F1/2 and by residues in IL2 , IL4 and IL5 in cluster G1/2 .', 'These fields extended into the headgroup region of the lipid bilayer , therefore pre-disposing these conformations for interactions with negatively-charged lipid such as phosphatidylserine or PIP2 .'], ['The physiological role of the hDAT is to rapidly remove the previously released dopamine by uptake in the pre-synaptic neuron , which leads to a fast drop of dopamine concentration .', 'Oligomerization of human monoamine transporters is involved in transporter function at multiple levels: surface expression requires transporter dimerization to pass quality control at the ER exit sites for loading into COPII vesicles [41] .', 'Transporter function seems to be modulated by oligomerization , including substrate transport activity and amphetamine-elicited neurotransmitter efflux by reverse transport through the transporter [19 , 23 , 24] as well as endocytosis from the plasma membrane [42] .', 'Also , some evidence of cooperativity between transporter protomers has been reported in the literature [22 , 25 , 43] .', 'Early studies on transporter oligomerization have identified C243 and C306 [13 , 14] in hDAT to be involved in oligomer formation .', 'These two residues are positioned at opposite sites on the transporter surface , i . e . C243 is located within TMH4 , while C306 is found towards the end of EL3 .', 'It was therefore conceivable that large oligomer structures of theoretically infinite size could form , if both elements are involved .', 'Unexpectedly , only dimer , trimer and tetramers of hDAT were identified after chemical cross-linking using Cu2+ or Cu2+-phenanthroline as oxidizing agent .', 'Interestingly , the C243 cross-link was formed only to a limited extent , in contrast to the C306 cross-link .', 'These data on human DAT are fully consistent with our results: We found a symmetric cluster of conformations , in which two C306 residues were pre-disposed for cross-link formation , but we were not able to observe such clusters with residue C243 in the interface .', 'Notably , chemical cross-link formation is not necessarily representative of a conformational equilibrium situation , because it reports on reactivity , rather than on equilibrium distribution .', 'Thus , a dimer has to exist only for a time period that is long enough for the chemical reaction to take place .', 'hDAT is not chemically cross-linked at the plasma membrane under normal conditions , while under oxidizing conditions [13 , 14] the cross-linked dimers were a minor component .', 'This is important because we can infer that the C243 dimer should be very rare , while the C306 dimer should be of low abundance , albeit higher than the C243-C306 heterodimer species .', 'Such low frequency is precisely what we observed in our dataset .', 'TMH11 and TMH12 have been observed to be within a dimer interface in hSERT [17] .', 'The similarity of hSERT and hDAT in many functional and biophysical aspects suggests that such interfaces may exist in hDAT as well .', 'We identified TMH11 in the interface of heterodimers , whereas the involvement of TMH12 appears to be more complex .', 'Of all the transmembrane helices among the SLC6 transporter family , only the conformation of TMH12 is not conserved between the bacterial homolog LeuT and human transporters [7–9] .', 'This is attributable to a prominent kink in the center of the membrane .', 'The dimeric LeuT crystal structure showed TMH9/12 at its interface .', 'However , the kink in TMH12 makes the same dimer geometry virtually impossible .', 'We still find TMH12 in the dimer interface , nonetheless the interface is limited to residues located predominantly towards the extracellular and possibly intracellular ends .', 'Sequence analyses carried out before solving the first LeuT structure uncovered a putative leucine heptad repeat in TMH2 , which was suggested to be involved in transporter oligomerization of rGAT1 [44] and hDAT [21] .', 'Surface targeting of both transporters was largely reduced and FRET analysis established a loss of oligomer formation in rGAT1 following mutagenesis of the respective leucine side chains to alanine .', 'However , the crystal structures of LeuT , dDAT and hSERT did not confirm the existence of the predicted leucine heptad repeat [45] .', 'A π-helix element in the middle of TMH2 creates a frame shift in the positioning of the helix residues with respect to a canonical α-helix .', 'Hence , the leucine residues can no longer be aligned to form a heptad repeat .', 'Furthermore , two of the four residues are not surface-exposed and cannot be involved in protein-protein interactions .', 'The C-terminal helix , directly following TMH12 , is absolutely essential for the surface expression of hDAT and the related transporters [32 , 42 , 46 , 47] , since it plays a key role in protein folding and trafficking , as well as in the interaction with the transporter core [48 , 49] .', 'Moreover , a motif located in the C-terminal region is necessary for recognition by the SEC24C vs . SEC24D components of the ER export ( COPII ) machinery [47 , 50] .', 'In addition , the C-terminus harbors the FREK sequence ( residues 587–590 ) , which is the binding site for the small ras-like GTPase Rin1 , that is involved in PKC-mediated endocystosis of the transporter [51 , 52] .', 'Cluster D revealed dimers which interacted only through the C-terminal helices , stabilized by a salt bridge and aromatic interactions between the protomers , but devoid of contacts with the transmembrane region .', 'The C-terminal helix was embedded in the lipid headgroup region of the membrane , thereby affecting the lipid bilayer .', 'This is indicative of a general biophysical property , which can possibly be adopted by every member of the SLC6 transporter family , and is also consistent with the specificity of SEC24 isoforms required for ER export of individual proteins .', 'Namely , there are four human SEC24 isoforms ( SEC24A-D ) that recognize and bind some ~6000 membrane proteins encoded for in the human genome .', 'The entire ensemble revealed that we obtained a set of structures that clustered into 8 dimer conformations , consisting of four symmetric and four asymmetric dimers , showing in total 6 partially overlapping and consequently mutually-exclusive interfaces .', 'Mapping the entire ensemble revealed that the putative interfaces covered a large part of the hDAT surface: it consisted of the scaffold domain comprising TMH 4 , 9 , but also TMH 3 , 8 , and 12 at their extracellular or intracellular ends .', 'In addition , TMH 5 and 11 , which are diagonally oriented at the transporter surface , extensively contribute to the dimer interfaces .', 'It is remarkable that the four helices of the bundle domain ( TMH 1 , 2 , 6 and 7 ) appear to be absent from the dimer interface , with the exception of the first turn of TMH6 until residue W311 , which formed contacts in some dimers .', 'Analysis of all trajectories revealed that on rare occasions ( a total of 7 trajectories ) , transient dimers formed with the bundle domain as the core of the dimer interface .', 'However , they were always unstable and separated within 0 . 5 μs .', 'The lack of such dimers was not due to limited sampling , because from our ensemble of 512 independent simulations , we would have expected 20–40 dimers forming through random encounters .', 'Importantly , the PMF profiles of these seven dimers were flat ( Fig 6I ) , verifying the lack of dimer-stabilizing interactions .', 'Collectively , these data suggest that hDAT establishes a force that opposes dimer formation at its bundle domain surface .', 'Our data indicate that the bias is , at least , two-fold:', 'i ) hDAT seems to perturb the membrane to make encounters less likely .', 'ii ) Interactions of residues in the bundle domain with a second hDAT transporter are generally weak , because EL3 forms a rim-like structure at the transporter-lipid interface and is thereby floating in the membrane headgroup region , strongly limiting the extent of the transporter surface in direct contact with the dimer interface .', 'Biological membranes are a complex mixture of many types of lipids , asymmetrically and non-homogeneously distributed over the two leaflets .', 'A full description of this complexity would be desirable , but is not yet achievable .', 'The cholesterol content in the ER membrane is below 5% , and it is devoid of PIP2 .', 'Oligomerization of the homolog hSERT was shown to be indistinguishable between the ER membrane and a PIP2 free plasma membrane , suggesting that transporter oligomerization is largely insensitive to the membrane composition .', 'The use of a POPC lipid membrane is therefore a good compromise , as it consists of the dominant phospholipid species in the plasma membrane as well as the ER membrane , though details might differ .', 'Not all membrane properties have the same importance for oligomerization .', 'Experi
['Introduction', 'Results', 'Discussion', 'Conclusion', 'Materials and methods']
['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 .']
['The human dopamine transporter efficiently removes the neurotransmitter dopamine from the synaptic cleft .', 'Alteration of dopamine transporter function is associated with several neurological diseases , including mood disorders or attention-deficit hyperactivity disorder , but is also a major player in addiction and drug abuse .', 'Functional studies have revealed that not only is transporter oligomerization involved in surface expression and endocytosis , but , more importantly , in reverse transport ( efflux ) of dopamine that is triggered by amphetamine-like drugs of abuse .', 'Structural knowledge of transporter oligomerization is largely missing .', 'We performed a large scale comprehensive computational study on transporter oligomerization to reveal dimer geometries and the residues involved in the interfaces .', 'The dimer conformations we find in our dataset are fully consistent with all available experimental data in the literature , but also show novel interfaces .', 'We further verified all dimer geometries by free energy calculations .', 'Our results identified an unpredicted—but for the mechanism of substrate transport essential—property: the bundle domain , which moves during the transport cycle , is excluded from contributing to dimer interfaces , thereby allowing for unrestrained movements necessary to translocate substrates through the membrane .']
['dimers (chemical physics)', 'crystal structure', 'dopamine transporters', 'condensed matter physics', 'materials science', 'crystallography', 'oligomers', 'cellular structures and organelles', 'materials by structure', 'chemical properties', 'physical chemistry', 'lipids', 'solid state physics', 'dimerization', 'proteins', 'chemistry', 'cell membranes', 'physics', 'biochemistry', 'biochemical simulations', 'cell biology', 'biology and life sciences', 'physical sciences', 'computational biology', 'chemical physics']