Fig. 2: Comparison with experimental data and changes in protein flexibility and secondary structures induced by phosphorylation.

In the following, p1OPTN and p5OPTN refer to the case of the mono-phosphorylated and multiple phosphorylated variants, respectively. A For each replicate of the LC3B_1–119-p5OPTN_169–185 construct (n = 100,000 frames), we estimated a reduced χ² (χ²_red) over the simulation time. This value provides a measurement of the agreement between the experimental chemical shifts from NMR and the calculated chemical shifts from the MD trajectories. The metric is calculated for each atom type. Low values of χ²_red indicate better agreement between experiments and simulations. We reported the example of the MD replicate 1 of LC3B_1–119-p5OPTN_169–185 for the sake of clarity. The results for the other replicates are available in the OSF repository⁶⁰ as detailed in the Materials and Methods. B, C The average Cα RMSF for each replicate (replicate 1: continuous line; replicate 2: dashed-line; replicate 3: square-dot line), calculated using 100-ns time windows (n = 10 independent values) is shown for LC3B_1–120-p5OPTN_158–191 (B) and LC3B_1–120-p1OPTN_158–191 (C) compared to the unphosphorylated construct, respectively. D, E The bar plots show the secondary structure content according to the DSSP dictionary using a concatenated trajectory with all the MD replicates for LC3B_1–120-OPTN_158–191 (D) and LC3B_1–120-p5OPTN_158–191 (E) (n = 300,000 frames for each system). It can be observed that the multiple phosphorylation slightly rearranges the propensity to β-strand formation while also rearranging the α-helix content in OPTN. F The panel shows the mono-dimensional free energy surface (FES) profiles for the parabeta collective variable employed in the metadynamic simulations (n = 506 frames) to describe the intermolecular β–sheet between OPTN and LC3B, i.e., measuring the number of segments that resemble a parallel β-sheet conformation. The wild-type LC3B_1–120-OPTN_158–-191 and penta-phosphorylated LC3B_1–120-p5OPTN_158–191 are highlighted in green and red, respectively. We observe that the phosphorylation induces an increase in intermolecular β-sheet formation.