Computational modeling of the N-terminus of the human dopamine transporter and its interaction with PIP₂ -containing membranes

Abstract

The dopamine transporter (DAT) is a transmembrane protein belonging to the family of Neurotransmitter:Sodium Symporters (NSS). Members of the NSS are responsible for the clearance of neurotransmitters from the synaptic cleft, and for their translocation back into the presynaptic nerve terminal. The DAT contains long intracellular N- and C-terminal domains that are strongly implicated in the transporter function. The N-terminus (N-term), in particular, regulates the reverse transport (efflux) of the substrate through DAT. Currently, the molecular mechanisms of the efflux remain elusive in large part due to lack of structural information on the N-terminal segment. Here we report a computational model of the N-term of the human DAT (hDAT), obtained through an ab initio structure prediction, in combination with extensive atomistic molecular dynamics (MD) simulations in the context of a lipid membrane. Our analysis reveals that whereas the N-term is a highly dynamic domain, it contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer (totaling >2.2 µs). Combining MD simulations with continuum mean-field modeling we found that the N-term engages with lipid membranes through electrostatic interactions with the charged lipids PIP2 (phosphatidylinositol 4,5-Biphosphate) or PS (phosphatidylserine) that are present in these bilayers. We identify specific motifs along the N-term implicated in such interactions and show that differential modes of N-term/membrane association result in differential positioning of the structured segments on the membrane surface. These results will inform future structure-based studies that will elucidate the mechanistic role of the N-term in DAT function.

Combined in vitro and in silico approaches to the assessment of stimulant properties of novel psychoactive substances – The case of the benzofuran 5-MAPB

“…We present here, on the basis of the crystal structure from Drosophila melanogaster dopamine transporter (dDAT) (PDB ID: 4XP1) (Wang et al, 2015), a molecular model of 5-MAPB bound to rat DAT (rDAT), in comparison with that of dopamine, amphetamine, 5-APB, MDMA, cocaine and RTI-121 also bound to rDAT. Our studies compare the structural changes produced by various ligands binding to rDAT to the already ongoing work in hDAT models (Hamilton et al, 2013;Hansen et al, 2014;Khelashvili et al, 2015aKhelashvili et al, , 2015b since it is the primary animal model used in functional studies and pre-clinical drug abuse testing.…”

“…The construction of the homology model of rDAT (rDAT dDAT ) in complex with dopamine uses established protocols previously described for the construction of a human DAT (hDAT) model (Hamilton et al, 2013;Hansen et al, 2014;Khelashvili et al, 2015aKhelashvili et al, , 2015b. Briefly, as the template, the homology model uses the recent crystal structure of the neurotransmitter sodium symporter (NSS) DAT from Drosophila melanogaster (dDAT) bound to dopamine (PDB ID: 4XP1) (Wang et al, 2015).…”

“…', n.d.). Dopamine was then re-docked into this prepared model of rDAT dDAT as described earlier (Khelashvili et al, 2015a(Khelashvili et al, , 2015b.…”

“…cocaine, amphetamine and RTI-121 were prepared using established protocols (Khelashvili et al, 2015a(Khelashvili et al, , 2015b.…”

“…A previous work (Beuming et al, Nature 2008) on a homology model of human DAT based on is not disrupted similar to our initial docks. It is important to note, as mentioned above, that the previous model of hDAT was based on a template of LeuT (Beuming et al, 2008) while our current model of rDAT is based on dDAT, and includes both N-and C-terminal regions, constructed from a previous protocol developed for hDAT (Khelashvili et al, 2015a).…”