The dopamine transporter (DAT) is a transmembrane protein belonging to the family of Neurotransmitter:Sodium Symporters (NSS). 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 usually 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 Berbamine 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 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 around the membrane surface. These results will inform future structure-based studies that will elucidate the mechanistic role of the N-term in DAT function. (dDAT) was reported recently 7 and revealed 12 TM segments a domain architecture that has been predicted as well from previous molecular modeling of human DAT (hDAT) based on sequence homology to the bacterial Leucine transporter (LeuT) 8-14 for which several crystal structures have existed since 200515-22. But in contrast to LeuT DAT has much longer cytoplasmic N- and C-terminal segments. These intracellular segments possess numerous putative phosphorylation sites and several protein kinases have been implicated in the regulation of DAT function 23-26. The phosphorylation of the N-terminal segment at serine residues positioned in its distal portions (i.e. close to its starting residue Met1 see Physique 1A) leads to the intriguing phenotype of efflux in which the substrate DA is usually transported Rabbit Polyclonal to USP15. via DAT in the reverse direction out of the cell 27-31. Under physiological conditions efflux can be triggered by the action of the psychostimulant amphetamine (AMPH) which apparently leads to a DAT conformation suitable for the phosphorylation of the N-terminus. Indeed in studies of phosphomimetic S-to-D mutations substituting the distal serine residues of the N-terminus (N-term) DAT-mediated DA efflux was observed even in the absence of AMPH 27. Physique 1 ((bottom row) DAT N-terminal segments illustrating low Berbamine sequence conservation between the two species. The conserved residues are colored. The alignment has been performed with the BLAST tool74. ( … Interestingly efflux can be regulated separately from the substrate uptake process. Thus we have shown that for hDAT charge-neutralizing substitution of K3 and K5 residues in the N-term with either Ala or Asn dramatically reduces AMPH-induced DA efflux while leaving the DA uptake unchanged 32. The same studies have suggested a central mechanistic role for direct binding of the N-term to highly charged anionic PIP2 (phosphatidylinositol 4 5 lipids in the efflux process 32. The importance of the efflux process is usually underscored by results from recent studies showing that it is affected by specific mutations in DAT linked to various neurological disorders 33-38. Despite rapid progress in identifying key elements of the molecular machinery that regulates the efflux process in NSS proteins (e.g. involvement of specific components of the cell membrane interactions with scaffolding proteins) 39-42 the molecular mechanism of DAT-mediated reverse transport remains unclear. This includes mechanistic questions about the role of the N-terminal segment (residues 1-59 in hDAT) for which structural information is usually lacking because this functionally important region in DAT is usually absent in the prototypical NSS the bacterial transporter LeuT and also had to be excised from the construct used to obtain the only available X-ray structure of the DAT protein from (dDAT) 7. Moreover the sequence of the DAT N-terminus is not homologous to any Berbamine protein with known Berbamine fold Berbamine and exhibits important variations among DAT proteins from different species (Physique 1A). To overcome this difficulty we sought a prediction of the three-dimensional (3D) conformation of the N-terminus from the human DAT (hDAT) by combining as described here structure prediction tools and extensive atomistic molecular dynamics (MD) simulations. The modeling carried out with the Rosetta software 43 yielded predictions of structured regions within the first 57 residues of the N-term..