the leubat mutants were expressed and purified as previously described except that lauryl maltose neopentyl glycol was used for solubilization and purification . after final purification by size - exclusion chromatography in -og , leubat was concentrated to 2.5 mg / ml and supplemented with saturated serotonin or mazindol . all leubat - drug complexes except mazindol were formed by soaking leubat crystals in crystallization solutions containing 3 - 20 mm of each drug . the structures were solved by molecular replacement using leut - trp structure as a search probe and then subjected to crystallographic refinement . functions of leubat mutants were examined by [ h]-paroxetine , [ h]-sertraline and [ h]-mazindol saturation and competition binding assays .
summarythe biogenic amine transporters ( bats ) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents that include selective serotonin reuptake inhibitors ( ssris ) , serotonin - norepinephrine reuptake inhibitors ( snris ) and tricyclic antidepressants ( tcas)1 , 2 . because eukaryotic bats are recalcitrant to crystallographic analysis , our understanding of the mechanism of these inhibitors and antidepressants is limited . 
 leut is a bacterial homolog of bats and has proven a valuable paradigm for understanding relationships between structure and function in bats3 . 
 however , because leut has only ~20% amino acid sequence identity to bats and is a promiscuous amino acid transporter4 , it does not recapitulate the pharmacological properties of bats . indeed , ssris and tcas bind in the extracellular vestibule of leut5 - 7 and act as non - competitive inhibitors of transport5 . 
 in contrast , multiple studies demonstrate that both tcas and ssris are competitive inhibitors for eukaryotic bats and bind to the primary binding pocket8 - 16 . here , we engineered leut to harbor human bat - like pharmacology by mutating key residues around the primary binding pocket . 
 the final leubat mutant binds the ssri sertraline with a binding constant of 18 nm and displays high affinity binding to a range of ssris , snris and a tca . 
 we determined 12 crystal structures of leubat in complex with four classes of antidepressants . 
 the chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle tm3 , wedge between tm3/tm8 and tm1/tm6 , and lock the transporter in a sodium and chloride - bound outward facing open conformation . together , these studies define common and simple principles for the action of ssris , snris and tcas on bats .