Conformational Flexibility Enables Function of a BECN1 Region Essential for Starvation‐Mediated Autophagy

Abstract

BECN1 is essential for autophagy, a critical eukaryotic cellular homeostasis pathway. Here we delineate a highly conserved BECN1 domain (residues 141–171), located between the previously characterized BH3 and coiled‐coil domains, and elucidate its structure and role in autophagy. The 2.0 Å Sulfur‐Single Wavelength Anomalous Diffraction crystal structure of this domain demonstrates that its N‐terminal half is unstructured, while its C‐terminal half is helical, hence we name it the Flexible Helical Domain (FHD). Circular dichroism, Double‐Electron‐Electron Resonance‐Electron Paramagnetic Resonance and Small Angle X‐ray Scattering (SAXS) analyses confirm that this domain is partially disordered even in the context of adjacent BECN1 domains. Molecular dynamic simulations fitted to SAXS data indicate that the FHD transiently samples more helical conformations in solution. Strikingly, the FHD is predicted to fold upon binding, and we find it becomes more helical in the presence 2,2,2‐trifluoroethanol, indicating it may undergo a disorder‐to‐helix conformational change upon binding to a partner. A completely helical conformation would place highly conserved FHD residues on the same face of the helix, suggesting that these residues may be involved in binding. Lastly, cellular studies show that highly conserved FHD residues are essential for the starvation‐induced up‐regulation of autophagy, the first BECN1 region shown to be critical for starvation‐induced autophagy rather than basal autophagy. Thus, our combined data suggest that the FHD likely undergoes a disorder‐to‐helix transition upon binding to appropriate partners, and conserved residues critical for this interaction are essential for starvation‐induced up‐regulation of autophagy.Support or Funding InformationThis work was supported by National Institutes of Health grants RO3 NS090939 (S.S.) and R15 GM113227 (C.C.); a National Science Foundation grant MCB‐1413525 (S.S.); North Dakota EPSCoR doctoral dissertation award for Y.M. (S.S.). AR was supported by a ORNL Laboratory Director's Research and Development SEED proposal 7278.