Chemical and structural investigation of the paroxetine-human serotonin transporter complex

Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand the binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation. Such modifications to SERT have led to questions about the relationships of these structures to the bona fide conformation and inhibitor binding poses of the wild-type transporter. To address these concerns, we characterized wild- type SERT with truncated N- and C-termini and thermostabilized variants of SERT bound with paroxetine using x-ray crystallography, single particle cryo-EM and biochemical techniques. Moreover, using a C-H functionalization approach to synthesize enantiopure analogues, we replaced the halide of the fluorophenyl group in paroxetine with either bromine or iodine. We then exploited the anomalous scattering of Br and I to define the pose of the respective paroxetine analog. These structures provide mutually consistent insights into how paroxetine and its analogs bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.