Structural Determinants of the Dopamine Transporter Regulation Mediated by G Proteins

Dopamine clearance in the brain is controlled by the dopamine transporter (DAT), a protein residing in the plasma membrane, which drives reuptake of extracellular dopamine into presynaptic neurons. Studies have revealed that the beta gamma subunits of heterotrimeric G proteins modulate DAT function through a physical association with the C-terminal region of the transporter. Regulation of neurotransmitter transporters by G beta gamma subunits is unprecedented in the literature; therefore, it is interesting to investigate the structural details of this particular protein-protein interaction. Here, we refined the crystal structure of the Drosophila melanogaster DAT (dDAT), modeling de novo the N- and C-terminal domains; subsequently, we used the full-length dDAT structure to generate a comparative model of human DAT (hDAT). Both proteins were assembled with G beta 1 gamma 2 subunits employing protein-protein docking, and subsequent molecular dynamics simulations were run to identify the specific interactions governing the formation of the hDAT:G beta gamma and dDAT:G beta gamma complexes. A [L/F]R[Q/E]R sequence motif containing the residues R588 in hDAT and R587 in dDAT was found as key to bind the G beta gamma subunits through electrostatic interactions with a cluster of negatively charged residues located at the top face of the G beta subunit. Alterations of DAT function have been associated with multiple devastating neuropathological conditions; therefore, this work represents a step toward better understanding DAT regulation by signaling proteins, allowing us to predict therapeutic target regions.