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. 2016 Apr 5;55(13):1945-58.
doi: 10.1021/acs.biochem.5b01264. Epub 2016 Mar 21.

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

Yang Mei  1 , Arvind Ramanathan  2 , Karen Glover  1 , Christopher Stanley  3 , Ruslan Sanishvili  4 , Srinivas Chakravarthy  5 , Zhongyu Yang  1 , Christopher L Colbert  1 , Sangita C Sinha  1
Affiliations

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

Yang Mei et al. Biochemistry. .

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.

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Figures

Figure 1
Figure 1
Sequence alignment of the BECN1 FHD from eight diverse eukaryotes. Residue numbers correspond to the human FHD. Yellow, orange and red backgrounds represent increasing sequence conservation with red corresponding to invariant residues. Secondary structure elements reported in this paper are displayed above the alignment with the orange line representing the disordered region and the orange cylinder representing the helical region. Residues predicted by the program ANCHOR (44) to nucleate folding upon binding are indicated. Arrows indicate residues that were mutated to Ala for cellular assays.
Figure 2
Figure 2
The FHD crystal structure. (A) The FHD monomer. The FHD backbone is displayed in orange ribbon, while highly conserved residues are labeled and rendered in stick, with atoms color-coded by type: C, orange; O, red; N, blue and S, yellow. This and all other molecular figures were made using PyMol. (B) The FHD trimer showing three molecules arranged around the crystallographic 3-fold are rendered as in (A). Highly conserved or structurally important residues from one monomer are labeled. (C) Electrostatic potential surface of the FHD trimer calculated using APBS. The figure on the left displays the face bearing the C-termini of the FHD monomers. The figure on the right displays the face bearing the N-termini of the FHD monomers. This face has a pocket that binds a sulfate molecule, rendered in stick and color-coded by atom type as in (A).
Figure 3
Figure 3
DEER-EPR data and data analysis on the spin labeled FHD. (A) The DEER signal intensity, F(t), as a function of time domain dipolar evolution (black curve). The data is analyzed by assuming that the distance distribution between the spin labels is a Gaussian function with the best fit shown by the red dotted curve. (B) The distance distribution function, P(r), derived from the analysis of the time domain signal in (A). The grey vertical bars indicate the approximate upper limit of reliable distances determined from DEER.
Figure 4
Figure 4
SAXS analysis of the BECN1 FHD. (A) Guinier Plot. (B) P(r) pairwise distribution. (C) Kratky plot.
Figure 5
Figure 5
MD conformer ensembles of the FHD fit to experimental SAXS data. The fits to the experimental SAXS data (red dots) are shown in dark grey and light grey for the MD simulations initiated from the part coil and part helical FHD trimer (left ensemble) and for the simulations initiated from the fully helical FHD trimer (right ensemble) respectively. An ensemble representation of conformers that show a good fit (0.5 ≤ χ2 ≤ 1.5) to the experimental SAXS data is shown for each set of simulations.
Figure 6
Figure 6
SAXS analysis of BECN1 FHD-CCD. (A) Guinier Plot. (B) P(r) pairwise distribution (C) Kratky plot.
Figure 7
Figure 7
Effects of TFE on the secondary structure content in various BECN1 constructs. CD spectra for the (A) FHD, (B) CCD and (C) FHD-CCD recorded at four different TFE v/v concentrations: 0%, 10%, 25% and 40%.
Figure 8
Figure 8
Effect of the mutation of conserved BECN1 residues on autophagy. (A) Light microscopy quantification of discrete GFP-LC3 puncta per cell in GFP-positive MCF7 cells co-transfected with GFP-LC3, WT or mutant BECN1 as indicated below the X-axis. Bars represent number of puncta per cell for each construct. (B) Representative images of GFP- LC3 staining in cells grown in starvation media and transfected with mutant BECN1 as indicated.
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