Fig. 2: Biophysical characterization of compound-LC3/GABARAP interactions. | Nature Communications

Fig. 2: Biophysical characterization of compound-LC3/GABARAP interactions.

From: Critical assessment of LC3/GABARAP ligands used for degrader development and ligandability of LC3/GABARAP binding pockets

Fig. 2

a Fluorescence polarization (FP) displacement assay titrations for compounds 1–13 measuring interaction with LC3B. Assays were run as technical replicates (n = 2) with data presented as mean values +/− SD of each data point. b FP data measuring the binding of Novobiocin to all six human LC3/GABARAP proteins using a p62 LIR-based tracer. Individual dose dependent titrations are depicted in Supplementary Fig. 2. Titrations were run as technical duplicates (n = 2) with data presented as mean values +/− SD of each data point. c Fluorescence polarization assay displacement curves for compound 1–4 against all LC3/GABARAPs against a p62 LIR-based tracer. Assays were run as technical duplicates (n = 2) with data presented as mean values +/− SD of each data point. d Interaction between LC3B and compounds 1–4 investigated by NMR. Representative fingerprint areas around the key K51 and V58 backbone HN resonances of 2D 1H‐15N correlation spectra for free LC3B (magenta) and LC3B containing control compound 12 (recorded at 700 MHz spectrometer as [1H-15N] fHSQC experiment) and compounds 1–4 (recorded at 800 MHz spectrometer as [15N,1H] BEST-TROSY) at 1:1 (yellow) and 1:2 (green) molar ratios are shown in overlay. Mapping of backbone HN resonances on LC3B sequence and structure are depicted in Supplementary Fig. 3. e Left plot: chemical shifts perturbations (CSP) values, induced by 12 at molar ratio 1:2, are plotted against LC3B residue numbers and mapped on 3D-structure (insert). The light green dashed line indicates the standard deviations (SD) over all residues, the orange dashed line indicates double SD values; residues with small (CSP < SD), intermediate (SD < CSP <2xSD) or strong (2xSD <CSP) CSP values are marked in grey, light green and orange, respectively. Other plots: compounds 1–3 induce insignificant CSP values at molar ratio 1:2, compound 4 induces small CSP around LC3B residues forming HP2 (right plot). Source data for (a, b, c, e) are provided as a Source Data file.

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