Fig. 1: Epitope transfer from dDAT facilitates GAT1 reconstruction.
From: Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism
a, X-ray structure of dDAT interacting with the Fab, 9D5 (PDB 4XP1), at the cytosolic face with heavy (blue) and light (pink) chain epitope interactions displayed as inset images. Interactions are given as dashed lines. b, Sequence alignment of the epitope region around IL3 and IL5 in dDAT compared with rGAT1WT. The epitope substitutions made in rGAT1WT to prepare the engineered construct, rGAT1EM, are highlighted in colored boxes. c, FSEC results displaying shifts in the GFP fluorescence peak elution volume in the complexed and uncomplexed forms of rGAT1WT and rGAT1Epi4 to highlight the presence of 9D5 interactions in the engineered rGAT1 construct. A.U., arbitrary units. d, 2D classes show the presence of Fab interactions with rGAT1EM in multiple orientations. e, 3H-GABA uptake assays were performed in two independent measurements (n = 2), each carried out in triplicate. Each point represents a mean of six measurements, and error bars represent s.e.m. The two independent datasets are displayed in Supplementary Figs. 4 and 5. The Km and Vmax values are 11 μM and 2,023 fmol per well per min for rGAT1WT and 4.2 μM and 855.7 fmol per well per min for rGAT1EM. f, Measurement of tiagabine and NO711 inhibition potency against rGAT1EM indicates an IC50 of 704 nM and 154.9 nM, respectively. Uptake measurements were performed as two independent experiments (n = 2), each carried out in triplicate. Each data point in the graph represents an average of all six measurements with error bars representing s.e.m. g, Binding measurements of rGAT1EM expressing membranes with NO711, a tiagabine analogue. The affinity (Kd) displayed by the interaction is 80 nM in comparison to 50 nM for rGAT1WT. Data points in the binding curve represent specific binding (means ± s.d.) calculated from total and non-specific binding measured by LC–MS shown in Supplementary Figs. 5–6 of one trial of four (n = 4) (rGAT1EM) independent experiments, each performed in triplicate, with error bars representing s.d.
