Insight into the differential binding mechanisms of substrate, cocaine, and therapeutic drugs with dopamine transporter based on molecular dynamics simulations.

The dopamine transporter (DAT) is a key membrane protein regulating dopaminergic signaling and a primary target of addictive drugs and therapeutic inhibitors. However, the atomic-level mechanisms by which endogenous substrates, addictive substances, and therapeutic inhibitors differentially regulate DAT conformation remain incompletely understood. Here, all-atom molecular dynamics (MD) simulations combined with MM/PBSA calculations, principal component analysis (PCA), and free energy landscape (FEL) analysis were employed to compare the binding modes of dopamine (DA), cocaine, Benztropine (BZT), and GBR12909 (GBR) to DAT. Three distinct binding modes were identified: DA is primarily electrostatic-driven, but this favorable contribution is largely offset by polar solvation penalty; cocaine and GBR rely on van der Waals and hydrophobic interactions; and BZT exhibits a dual-driver mode combining both strong electrostatic and van der Waals contributions. DA achieves recognition through an Asp12-mediated salt bridge and a multi-point hydrogen bond network, whereas cocaine and GBR bind via hydrophobic/aromatic residue embedding, and BZT through synergistic ionic anchoring and hydrophobic wrapping. Notably, Asp12 undergoes a functional role switch-favorable in DA and BZT, yet unfavorable in cocaine and GBR binding. Gate distance, PCA, and FEL analyses further revealed ligand-dependent gating regulation: BZT imposes the strongest conformational constraint, cocaine stabilizes the outward-open state, GBR seals the extracellular entrance with intracellular gate transition, and DA preserves conformational tunability. These findings provide a systematic atomic-level mechanistic framework for understanding DAT differential regulation and guiding the development of novel competitive inhibitors.