Fig. 4

S1014-AMBRA1 phosphorylation allows AMBRA1–LC3B binding. a ITC titrations of unmodified AMBRA1-LIR peptide (P0) compared with those of S1014-phosphorylated AMBRA1-LIR peptide (P1) into LC3B protein. The measurements were performed at 35 °C. Measured/estimated K_D values are indicated for each interaction. b Representative sections of HSQC spectra for ¹⁵N-labelled LC3B upon titration with P0 and P1. Both plots show fingerprint regions of the LC3B spectra (around HN resonance of K51). Molar ratios of protein/peptide are rainbow coloured (1:0, 1:0.125, 1:0.25, 1:0.5, 1:1, 1:2, 1:4 and 1:8; from red to violet) for each titration step. Arrows indicate the CSP for K51 and V58 HN backbone resonances. c K_D values calculated for the LC3B residue I34 upon titration with P0 and P1. Original CSP values are shown as black squares and the resulting fit is given as a red line in each plot. The original HSQC areas around I34 HN resonance are shown as a box under fitted curves. d CSP values (Δδ) at the last titration stages for LC3B protein are plotted against residues numbers. The orange dashed line indicates the standard deviations (σ) over all residues within each dataset, and the red dashed line indicates double σ values. The CSP values mapped on the LC3B protein structure (ribbon diagrams, PDB ID 1UGM) are shown in the upper right corner. Residues with small (Δδ < σ), intermediate (σ < Δδ < 2σ) or strong (2σ < Δδ) CSPs are marked in grey, yellow and red, respectively. e The root mean square fluctuation (RMSF) per residue of AMBRA1 decreases after S1014 phosphorylation (blue, green lines) compared to wt (magenta, red lines) and control P62 (black line) and is persistent with CHARMM22* (black, green and magenta lines) and CHARMM27 (blue and red lines) force fields. Shaded area corresponds to 1 σ. f Superimposition of highly populated (>20%) electrostatic interactions (red bars, thickness indicates occurrence) over the LC3B/AMBRA1-LIR structure. Interactions identified between residues of S1014-phosphorylated AMBRA1-LIR (right panel) and LC3B are identified by spheres and reproduced on the wt (left panel). Similar patterns were seen for simulations using CHARMM22* (shown here) and CHARMM27 force fields