Fig. 5: Binding of the activating sAB LK1 increases the occupancy of the lowest FRET peak.

a SRE-luciferase assay for signaling of ADGRL3 in the presence of 1 µM purified sABs LK1 and LK3 presented as fold increase over empty vector. Data were presented as mean ± SD of three repeats (n = 3) for a representative of three independent experiments. ***p = 0.0001; vs. HBS buffer treatment; two-way ANOVA. b smFRET population histogram of Y795:A871 UAA sensor in the presence of 1 µM LK1 antibody. Three single Gaussian distributions (gray) were fitted to the histogram centered at 0.44, 0.55, and 0.65 with cumulative fit in green. c Transition density plots of Y795:A871 UAA sensor with 1 µM LK1. Dashed lines indicate the most frequent transitions. d smFRET population histogram of Y795:A871 UAA sensor pair in the presence of 1 µM LK3 antibody. Three single Gaussian distributions (gray) were fitted to the histogram centered at 0.44, 0.55, and 0.65. The orange line represents the cumulative fit. e Transition density plots of Y795:A871 UAA sensor pair with 1 µM LK3. Dashed lines indicate the most frequent transitions. f Percent change in the number of states occurring in individual traces in the presence of antibodies LK1 or LK3 versus the apo receptor. g smFRET population histograms of Y795:A871 UAA sensor pair in control conditions in the presence of 1 µM LK1 or 1 µM LK3. h Change in occupancy of the three FRET states after the addition of LK1 or LK3 compared to the apo state. i Change in the occurrence of different FRET states among traces that showed only one state during recording between apo and LK1 or LK3 condition. j Superimposition of low-resolution maps of ADGRL3 in complex with sABs LK3 (in yellow) and LK1 (in green) showing a shift in the HormR/GAIN domains position in relation to the micelle. b–i Data represent mean ± s.e.m., n = 3 from three independent biological replicates. Source data for panels a, b, d, f, g, h, i are provided as a Source Data file.