Abstract
Dopamine (DA) transporter (DAT) is a major target for psychostimulant drugs of abuse such as cocaine that competitively binds to DAT, inhibits DA reuptake, and consequently increases synaptic DA levels. In addition to the central binding site inside DAT, the available experimental evidence suggests the existence of alternative binding sites on DAT, but detection and characterization of these sites are challenging by experiments alone. Here, we integrate multiple computational approaches to probe the potential binding sites on the wild-type Drosophila melanogaster DAT and identify a new allosteric site that displays high affinity for cocaine. This site is located on the surface of DAT, and binding of cocaine is primarily dominated by interactions with hydrophobic residues surrounding the site. We show that cocaine binding to this new site allosterically reduces the binding of DA/cocaine to the central binding pocket, and simultaneous binding of two cocaine molecules to a single DAT seems infeasible. Furthermore, we find that binding of cocaine to this site stabilizes the conformation of DAT but alters the conformational population and thereby reduces the accessibility by DA, providing molecular insights into the inhibitory mechanism of cocaine. In addition, our results indicate that the conformations induced by cocaine binding to this site may be relevant to the oligomerization of DAT, highlighting a potential role of this new site in modulating the function of DAT.