An Orchestrated Interaction Network at the Binding Site of Human SERT Enables the Serotonin Occlusion and Import

Serotonin transporter (SERT) regulates serotonergic signals by reuptaking serotonin from the synaptic clefts back into the presynaptic neurons. The recent resolution of the serotonin–SERT complex in multiple conformational states outlined the complete serotonin import cycle. However, a detailed functional appreciation of SERT also involves deciphering the coupling between global structural changes in the transport cycle to the bound chemicals to be transported. By employing molecular dynamics (MD) simulations and free energy calculations in different ligand binding states, here, we reveal how serotonin binding to SERT initiates the global conformational changes essential for serotonin import. Only when serotonin is bound to the central binding site, wedged between transmembrane helices (TMs) 3 and 8, can the system form an interaction network that bridges the two helical domains of the protein, thereby promoting the closure of an extracellular hydrophobic gate and sealing the bound serotonin. To test the role of this hydrophobic gate closure, we designed a series of nonequilibrium MD simulations to steer the outward-facing ↔ occluded transition with different gating configurations. The difference in nonequilibrium work required to fuel the transition indicates that the transition is more likely to happen when the extracellular gate is closed. The transition is not promoted when the gate is open or when 5-HT moves away from TM3 and TM8 toward an alternate pose. Such a local–global coupling is likely shared by other monoamine transporters considering the conservation of all involved structural elements.