Conformational flexibility of BECN1: Essential to its key role in autophagy and beyond

Abstract

BECN1 (Beclin 1), a highly conserved eukaryotic protein, is a key regulator of autophagy, a cellular homeostasis pathway, and also participates in vacuolar protein sorting, endocytic trafficking, and apoptosis. BECN1 is important for embryonic development, the innate immune response, tumor suppression, and protection against neurodegenerative disorders, diabetes, and heart disease. BECN1 mediates autophagy as a core component of the class III phosphatidylinositol 3-kinase complexes. However, the exact mechanism by which it regulates the activity of these complexes, or mediates its other diverse functions is unclear. BECN1 interacts with several diverse protein partners, perhaps serving as a scaffold or interaction hub for autophagy. Based on extensive structural, biophysical and bioinformatics analyses, BECN1 consists of an intrinsically disordered region (IDR), which includes a BH3 homology domain (BH3D); a flexible helical domain (FHD); a coiled-coil domain (CCD); and a β-α-repeated autophagy-specific domain (BARAD). Each of these BECN1 domains mediates multiple diverse interactions that involve concomitant conformational changes. Thus, BECN1 conformational flexibility likely plays a key role in facilitating diverse protein interactions. Further, BECN1 conformation and interactions are also modulated by numerous post-translational modifications. A better structure-based understanding of the interplay between different BECN1 conformational and binding states, and the impact of post-translational modifications will be essential to elucidating the mechanism of its multiple biological roles.

Keywords: BECN1/Beclin 1/ATG6/VPS30; autophagy; class III phosphatidylinositol 3-kinase complexes; conformational flexibility; interaction hub; intrinsically disordered protein.

Figure 1

Sequence alignment of BECN1 orthologs from diverse organisms. Increasing background color intensity corresponds to increasing residue conservation with red corresponding to invariant residues. Experimentally determined secondary structure is displayed above the alignment, with cylinders representing helices, arrows representing strands and lines representing coil, color‐coded by domains as follows: IDR (black), BH3D (cyan), FHD (orange), CCD (magenta), and BARAD (green). Solid colors indicate natively folded stable structural elements, horizontal stripes indicate elements that may fold as part of two domains and diagonal stripes indicate binding‐induced secondary structure. Anchor regions are boxed in black. Red triangles indicate the human NES.

Figure 2

Domain architecture of BECN1 and selected interactions. Domains are as in Figure 1, with corresponding domain structures displayed in ribbon above the schematic. Two structurally characterized interactions are displayed below the schematic: (i) The BECN1 BH3D (cyan ribbon) bound to M11 (grey molecular surface) and (ii) The BECN1 CCD (magenta ribbon): ATG14 CCD (salmon ribbon) heterodimer is shown modeled into its SAXS‐derived molecular envelope. Yellow‐green boxes represent interacting proteins that up‐regulate autophagy while grey boxes represent interacting proteins that down‐regulate autophagy. All molecular figures were prepared using the program PyMOL.48

Figure 3

Mutually exclusive packing of the “overlap helix”. The overlap helix‐containing BARAD (PDB 4DDP, green ribbon) is superimposed onto the CCD (PDB 3Q8T, magenta ribbon). Residues of the overlap helix in each structure, as well as the partner CCD helix, are colored in alternating pink and green.

Figure 4

The BECN1 CCD dimers. (A) BECN1 CCD homodimer, (B) VPS30:VPS38 CCD heterodimer and (C) BECN1:ATG14 CCD heterodimer. The upper panel shows three dimers in ribbon with interface residues rendered in stick with atoms color‐coded by atom type: C, magenta for BECN1, wheat for ATG14, green for VPS38 and yellow for VPS30; O, red; N, blue and S, yellow. The length of each dimer is indicated. The lower panel shows the corresponding helical wheel of the dimers.

Figure 5

BECN1 interactions in the PI3KC3 complex II. All proteins are shown in ribbon, colored as: VPS30, yellow; VPS38, green; VPS15, grey, and VPS34, blue. Protein domains implicated in membrane interaction, the BECN1 BARAD, UVRAG BARA2 and PI3KC3 catalytic domain are labeled. Boxes indicate regions that were enlarged and rotated to demonstrate interactions. Interacting side chains, mutated to the VPS30 sequence from the 4.4 Å poly‐alanine structure, are displayed as sticks.

Figure 6

Displacement of the BECN1 BARAD domain because of the curved BECN1:ATG14 quaternary structure. All proteins are shown in ribbon, colored as in Figures 4 and 5. Arrows indicate altered positions of equivalent residues.