Fig. 3: Interactions of putative LC3 ligands measured by NMR and pull-down—MS based assays.

a Interaction between GABARAPL2 and compounds 1–4 investigated by NMR. Representative areas of GABARAPL2 [¹⁵N,¹H] BEST-TROSY (recorded at 600 MHz spectrometer for compounds 1–3 and 900 MHz spectrometer for 4) spectra around the key residues L50, I32 and Y106 backbone HN resonances are shown in overlay with free GABARAPL2 (magenta), and in presence of 1:1 (yellow) and 1:2 (green) molar ratio of each compound (indicated above each plot). Arrows in the plot for GABARAPL2:4 interaction show directions of large chemical shift perturbations for the resonances which are in the intermediate exchange mode and could not be tracked until the latest titration steps, indicating the strongest interaction of these residues with GABARAPL2. Mapping of backbone HN resonances on GABARAPL2 sequence and structure and additional NMR data analysis are depicted in Supplementary Fig. 5. b CSP values, induced by compounds 1–4 at molar ratio 1:2, are plotted against GABARAPL2 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. The blue color for sequence- and 3D-mapping for compound 4 are for GABARAPL2 residues which undergo strong intermediate exchange mode (significant decrease of the resonances intensity upon titration with (4). c Exemplary mass spectrometry data expressing a mass shift of LC3B after treatment with compound 7. Full data set for compounds 5–7 on all LC3/GABARAPs is depicted in Supplementary Fig. 6. d Chemoproteomic competition assays for target deconvolution of 8F20 (3) and 10O5 (4). Affinity matrices for pulldown experiments were synthesized by amide coupling yielding (18) and (19) attached to (NHS-activated) Sepharose beads. Competition experiments were performed with free compound 3 and PEG-linked compound 4 at nine concentrations and residual binding was calculated relative to a DMSO control. Of the over 4000 proteins identified, only KIF11 showed robust dose-dependent binding to 19 (EC₅₀ = 290 nM). Both the 18- and 19-based affinity matrix assays did not enrich for the reported targets LC3/GABARAP in HEK293T cell lysate. Source data for Fig. 3b, d are provided as a Source Data file.