Abstract
Serotonin is a ubiquitous chemical transmitter with particularly important roles in the gastrointestinal, cardiovascular and central nervous systems. Modulation of serotonergic signaling occurs, in part, by uptake of the transmitter by the serotonin transporter (SERT). In the brain, SERT is the target for numerous antidepressants including tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs). Despite the importance of SERT in human physiology, biochemical, biophysical and high-resolution structural studies have been hampered due to the instability of SERT in detergent micelles. To identify a human SERT (hSERT) construct suitable for detailed biochemical and structural studies, we developed an efficient thermostability screening protocol and rapidly screened 219 mutations for thermostabilization of hSERT in complex with the SSRI paroxetine. We discovered three mutations—Y110A, I291A and T439S –that, when combined into a single construct, deemed TS3, yielded a hSERT variant with an apparent melting temperature (Tm) 19°C greater than that of the wild-type transporter, albeit with a loss of transport activity. Further investigation yielded a double mutant—I291A and T439S—defined as TS2, with a 12°C increase in Tm and retention of robust transport activity. Both TS2 and TS3 were more stable in short-chain detergents in comparison to the wild-type transporter. This thermostability screening protocol, as well as the specific hSERT variants, will prove useful in studies of other integral membrane receptors and transporters and in the investigation of structure and function relationships in hSERT.
Highlights
The human serotonin transporter belongs to the family of neurotransmitter sodium symporters (NSSs) that terminate neurotransmission by clearing neurotransmitters from the synaptic cleft [1]. hSERT pumps serotonin from the synaptic cleft and into presynaptic neuronsPLOS ONE | DOI:10.1371/journal.pone.0145688 December 22, 2015Thermostabilization of Antidepressant-Bound hSERT
To identify positions that could thermostabilize hSERT, we performed sequence alignment of hSERT with Drosophila melanogaster dopamine transporter (dDAT) (PDB ID: 4M48, 49% sequence identity) and manually selected 219 point mutations at 160 different residues based on the following criteria: 1) large charged residues in highly charged clusters, especially those on the surface; 2) kinked regions adjacent to α-helices involved in conformational changes; 3) branched side chains in α-helices; 4) residues lining solvent-exposed cavities; 5) post-translationally modified residues; 6) residues that are different in leucine transporter (LeuT); 7) mutations known to thermostabilize rSERT and dDAT
To facilitate the rapid screening of hSERT mutants we set out to develop a highthroughput scintillation proximity assay (SPA)-based thermostability screen that can rapidly screen for constructs with enhanced thermostability in a ligand-bound conformation (Fig 2)
Summary
The human serotonin transporter (hSERT) belongs to the family of neurotransmitter sodium symporters (NSSs) that terminate neurotransmission by clearing neurotransmitters from the synaptic cleft [1]. hSERT pumps serotonin from the synaptic cleft and into presynaptic neurons. Though structure determination of eukaryotic membrane proteins continues to lag significantly behind other targets, the use of the lipidic cubic phase, new detergents, antibody fragments, T4 lysozyme fusions and thermostabilized constructs have increased the number of structures determined by X-ray crystallography [13,14,15,16]. Even with these technological breakthroughs, eukaryotic membrane protein crystallization and structure determination remains difficult as highlighted by the fact that
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