Decoding phospho-regulation and flanking regions in autophagy-associated short linear motifs

Area of Science:

• Molecular Biology
• Biophysics
• Computational Biology

Background:

• Short Linear Motifs (SLiMs) mediate protein-protein interactions, often involving intrinsically disordered proteins.
• Post-translational modifications, like phosphorylation, and flanking residues significantly influence SLiM function, but mechanisms remain unclear.
• LC3 Interacting Regions (LIRs) are critical SLiMs for autophagy, highlighting the need to understand their regulation.

Purpose of the Study:

• To elucidate the atomic-level mechanisms of phospho-regulation in SLiMs, specifically LIRs involved in autophagy.
• To investigate the interplay between phosphorylation and flanking regions in modulating the binding of the Optineurin LIR to LC3.
• To characterize the impact of disease-associated mutations on these phospho-regulated interactions.

Main Methods:

• Integration of biomolecular simulations and in silico high-throughput mutational scans.
• Application of biophysical experiments, including Microfluidic Diffusional Sizing and Surface Plasmon Resonance.
• Characterization of binding affinities, dissociation constants, and kinetics for SLiM variants.

Main Results:

• Specific phosphorylation events and flanking residues were shown to modulate Optineurin LIR binding to LC3 at the atomic level.
• Disease-associated mutations were found to alter these interactions, particularly in the phosphorylated context.
• A robust toolkit was established for characterizing phospho-regulated SLiMs, enabling precise affinity and kinetic measurements.

Conclusions:

• Phosphorylation and flanking residues are key regulators of LIR-LC3 interactions, with significant implications for autophagy.
• Understanding these regulatory mechanisms is crucial for deciphering cellular processes and the impact of disease mutations.
• The developed toolkit offers a versatile approach for studying phospho-regulated protein interactions.