Fig 1.
“Snake” plot showing mutated residues.
Residues in purple were mutated in the thermostability screen. Of the tested constructs three were combined to generate a thermostabilized hSERT construct Y110A, I291A and T439S (red). Transmembrane helices are labeled in black boxes and extracellular loops are labeled as EL2 and EL4. hSERT also has a disulfide bond and two predicted glycosylated asparagine residues, which are demarcated by a black line and green hexagons, respectively. Topology and position of the transmembrane helices were determined by sequence alignment to the structure of dDAT.
Fig 2.
a, HEK293S cells were transiently transfected with the plasmid containing the encoded hSERT-GFP-H10 fusion protein. After expression for two days, paroxetine was added and the cells were solubilized. The amount of bound [3H]paroxetine is measured using Cu-YSi SPA beads. The sample was repetitively heated with the amount of bound paroxetine measured at each step. Constructs with high relative amounts of bound paroxetine after heating represent thermostabilizing mutations. b, Scheme of SPA step. Prior to heating, hSERT was bound to the Cu-YSi beads via the H10 tag. Any bound [3H]paroxetine which is in close proximity to the bead which undergoes β-decay can cause a detectable scintillation event. After heating, the protein is denatured and unable to bind [3H]paroxetine. Unbound [3H]paroxetine is not close enough to the Cu-YSi bead to cause a scintillation event.
Fig 3.
Results from paroxetine-bound thermostability screening assay.
a, Comparison of maximum bound [3H]paroxetine and measured melting temperature (Tm). Dotted lines represent values for wild-type hSERT. The gray region represents mutants that have less than 10% of paroxetine binding compared to wild-type hSERT and also have large errors in Tm measurements. b, Curve fits for wild-type hSERT and the two most stabilizing mutants, Y110A and I291A. c, Comparison of Tm values from 96-well format single-point assay and traditional melting curve measurement. All CPM values are specific CPM. Error bars represent standard errors of the mean.
Fig 4.
[14C]Serotonin uptake measurements.
a, The [14C]serotonin uptake of single mutants and b, combined mutants. c, Michaelis-Menten plot for [14C]serotonin uptake of wild-type and TS2 mutant gave a KM of 3.4 and 5.8 μM, respectively. Error bars represent standard errors of the mean.
Fig 5.
Increased thermostability and detergent stability.
a, The TS2 and TS3 mutations increase the stability of hSERT by 12 and 19°C, respectively. b, Strategy for combining mutants. TS2 and TS3 were made to increase hSERT stability. c, TS3 mutations increase the stability of hSERT in a wide range of detergents (MNG: lauryl maltose neopentyl glycol, C12M: n-dodecyl-β-D-maltopyranoside, DM: n-decyl-β-D-maltopyranoside, C10thioM: n-decyl-β-D-thiomaltopyranoside, C9M: n-nonyl-β-D-maltopyranoside, C8M: n-octyl-β-D-maltopyranoside) Columns without bars indicate it was not possible to measure the thermostability, potentially due to stability below room temperature. Error bars represent standard errors of the mean.
Fig 6.
Paroxetine binding affinity of TS3 and wild-type hSERT.
The paroxetine binding affinity of wild-type hSERT and TS3 was measured by membrane filter binding. Total counts were fit to a one site model accounting for ligand depletion due to the high affinity of the ligand, and representative curves are shown. The paroxetine Kd was 0.14, 0.22 and 0.103 for wild-type hSERT, TS2 and TS3, respectively. Error bars represent standard errors of the mean.
Fig 7.
The position of hSERT mutations in the dDAT structure.
The position of the three characterized thermostabilizing mutations are shown superimposed on the homologous residues from the dopamine transporter structure (PDB ID: 4M48). The overall positions are shown from side (a) and top (b) views. c, Close up of view of Y110A (top), I291A (middle), and T439S (bottom) are shown on residues Y58, A275, and S422 from dDAT, respectively. Side chains are shown as black spheres, sodium atoms are magenta spheres, chloride atoms are green spheres, nortriptyline is cyan.