Fig. 5: High-throughput mutational scans.
From: Decoding phospho-regulation and flanking regions in autophagy-associated short linear motifs
A Binding isotherms of OPTN169–185 (1) pS177 (n = 5 independent experiments), (2) S177D (n = 3 independent experiments), (3) S177E (n = 3 independent experiments), and (4) WT (n = 3 independent experiments) FITC-labeled peptide variants to GST-LC3B by microfluidic diffusional sizing. Raw data from all technical replicates are shown as individual dots. Each biological replicate is fitted independently, and the resulting nonlinear curves are overlaid. The reported Kd, Rh_free, and Rh_complex values represent the mean ± standard deviation across the biological replicates, with each value obtained from a separate fit. R² values indicate the goodness of fit for each individual curve. B–D The heatmaps for the saturation scan in silico for LC3B1–120-OPTN158–191 (B), LC3B1–120-p5OPTN158–191 (C), and LC3B1–120-p1OPTN158–191 (D) are illustrated with a truncation of the ΔΔG values at 3 kcal/mol for visualization purposes. We can observe that the pentaphosphorylation activates binding hotspots in the N-terminal flanking region for the binding to LC3B (B, C). The effect cannot be recapitulated by the single phosphorylation (C, D). E, F The dotplot summarizes the results of the MAVISp assessment in the simple (E) and ensemble (F) modes. Black, white, and gray dots represent mutations predicted with a damaging, neutral, or uncertain effect for one of the MAVISp modules, respectively.
