title: Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram.
abstract: The serotonin transporter is a sodium and chloride-coupled transporter that "pumps" extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI-cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 Å resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.
authors.affiliation: Vollum Institute, Oregon Health & Science University.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Coleman
authors.affiliation: Graduate Group in Biophysics, University of California, San Francisco.
authors.forename: Evan M
authors.initials: EM
authors.lastname: Green
authors.affiliation: Vollum Institute, Oregon Health & Science University; Howard Hughes Medical Institute, Oregon Health & Science University; gouauxe@ohsu.edu.
authors.forename: Eric
authors.initials: E
authors.lastname: Gouaux
mesh_terms: D015283:Citalopram / Q000378:metabolism*; D003460:Crystallization; D006801:Humans; D011485:Protein Binding; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017367:Selective Serotonin Reuptake Inhibitors
chemical_list: D015283:Citalopram; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; C493230:SLC6A4 protein, human