Abstract
BECN1 is essential for autophagy, a critical eukaryotic cellular homeostasis pathway. Here we delineate a highly conserved BECN1 domain located between previously characterized BH3 and coiled-coil domains and elucidate its structure and role in autophagy. The 2.0 Å sulfur-single-wavelength anomalous dispersion X-ray 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 spectroscopy, double electron-electron resonance-electron paramagnetic resonance, and small-angle X-ray scattering (SAXS) analyses confirm that the FHD 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. FHD helicity increases in 2,2,2-trifluoroethanol, suggesting it may become more helical upon binding. Lastly, cellular studies show that conserved FHD residues are required for starvation-induced autophagy. Thus, the FHD likely undergoes a binding-associated disorder-to-helix transition, and conserved residues critical for this interaction are essential for starvation-induced autophagy.
Original language | English |
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Pages (from-to) | 1945-1958 |
Number of pages | 14 |
Journal | Biochemistry |
Volume | 55 |
Issue number | 13 |
DOIs | |
State | Published - Apr 19 2016 |
Funding
This work was supported by National Institutes of Health Grants RO3 NS090939 (S.C.S.) and R15 GM113227 (C.L.C.), National Science Foundation Grant MCB-1413525 (S.C.S.), a North Dakota EPSCoR doctoral dissertation award for Y.M. (S.C.S.), and a Laboratory Director's Research and Development SEED proposal 7278 (A.R.). We also acknowledge the North Dakota State University (NDSU) Core Biology Facility (funded by National Institutes of Health Grant P30 GM103332-01) for access to tissue culture facilities and the NDSU Advanced Imaging and Microscopy Core Laboratory for access to microscopy and imaging equipment. Work performed at Bio-CAT was supported by National Institute of General Medical Sciences Grant 9P41 GM103622. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the APS, a U.S. Department of Energy Office (DOE) of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Oak Ridge National Laboratory (ORNL) is operated by UT-Battelle, LLC, for the DOE under Contract DE-AC05-00OR22725. This research used resources of the Oak Ridge Leadership Computing Facility at ORNL.