Mechanistic Characterization of the Allosteric Communications between the Central Binding Site and the Extracellular Vestibule of the Serotonin Transporter

Abstract

The serotonin transporter (SERT) is a primary target for antidepressants, e.g. S-citalopram and imipramine to alleviate symptoms of depression and anxiety, and functions by exploiting pre-existing ion gradients of Na+, Cl−, and K+ to translocate serotonin from the synaptic cleft into the presynaptic neuron. SERT crystal structures reveal that two S-citalopram molecules bind in both the central binding (S1) site and a site in the extracellular vestibule (S2 site). We investigated the allosteric communications between the S1 and S2 sites by carrying out extensive molecular dynamics (MD) simulations of hSERT in complex with various combinations of S1- and S2-bound ligands. The comparative analysis of the simulation results in combination with in vitro binding assays and mutagenesis reveal that the binding of S-citalopram or imipramine to the S1 site is allosterically coupled to the S-citalopram binding to S2 through the Thr497-Phe335 motif. The different configurations of this motif in turn affect the propensity of Glu494 in the S2 site to form a salt bridge with the S2-bound S-citalopram. In addition, our MD simulation results indicate that Lu AF60097, the first high-affinity S2-ligand, allosterically couples the ligand binding to S1 through a similar mechanism. In the presence of S1-bound imipramine but not S-citalopram, our combined in silico and in vitro analysis show that Lu AF60097 occupies a dynamically formed sub-pocket in the extracellular vestibule. We propose that the occupation of this cavity is responsible for the significantly improved affinity of Lu AF60097 in the presence of S1-bound imipramine. Together we reveal a coupling mechanism between the S1 and S2 sites that will facilitate rational development of high-affinity SERT allosteric inhibitors.