Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy

Abstract

Autophagy, an essential eukaryotic homeostasis pathway, enables sequestration of unwanted, damaged or harmful cytoplasmic components in vesicles called autophagosomes, enabling subsequent lysosomal degradation and nutrient recycling. Autophagosome nucleation is mediated by Class III phosphatidylinositol 3-kinase complexes that include two key autophagy proteins, BECN1/Beclin 1 and ATG14/BARKOR, which form parallel heterodimers via their coiled-coil domains (CCDs). Here we present the 1.46 Å X-ray crystal structure of the anti-parallel, human BECN1 CCD homodimer, which represents BECN1 oligomerization outside the autophagosome nucleation complex. We use circular dichroism and small-angle X-ray scattering (SAXS) to show that the ATG14 CCD is significantly disordered, but becomes more helical in the BECN1:ATG14 heterodimer, although it is less well-folded than the BECN1 CCD homodimer. SAXS also indicates that the BECN1:ATG14 heterodimer is more curved than other BECN1-containing CCD dimers, which has important implications for the structure of the autophagosome nucleation complex. A model of the BECN1:ATG14 CCD heterodimer that agrees well with the SAXS data shows that BECN1 residues at the homodimer interface are also responsible for homodimerization, enabling us to identify ATG14 interface residues. Lastly, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on coimmunoprecipitation of the partner, and demonstrate that these mutations abrogate starvation-induced up-regulation of autophagy, but do not impact basal autophagy. Thus, this research provides insights into structures of the BECN1 CCD homodimer and the BECN1:ATG14 CCD heterodimer, and identifies interface residues important for BECN1:ATG14 heterodimerization and for autophagy.

The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity

“…12,13 To investigate whether the BARA domain could contribute to dimerization, we repeated the SEC-MALS experiments with the construct BECN 1-265 . SEC-MALS analysis at 100 μ M shows an asymmetric peak with an average mass of 40.1 kDa (Figure 5A).…”

“…We also note that the homodimer K D for the BECN 1-265 construct is 35-fold tighter than the value for the isolated human CC domain. 13 Our BECN 1-265 construct contains the entire CC domain used in their study, and we attribute this difference to the experimental methodology. During SEC-MALS experiments, the BARA construct eluted as a predominantly monomeric species in parallel experiments up to 100 μ M, but without an observable migration of the elution volume (Figure 5B and Figure S4C).…”

“…13,14,52 Moreover, this helix is the C-terminal portion of the CC domain and forms intramolecular contacts with a hydrophobic pocket of the BARA domain (Figure 6A). This interaction was first observed in the crystal structure of the BARA domain, which begins at residue 248, and shows this helix folded back on the BARA domain (Figure 6B).…”

“…Full-length BECN1 exhibits a propensity to form the homodimer ( K D ~ 0.45 μ M) that is stronger than that of the isolated CC domain (48 μ M). 13 We also compare the full-length protein to two truncated forms of BECN1 (Figure 1A): (1) the BARA domain (residues 248–450) and (2) a BARA-less construct that contains the complete set of domains that are N-terminal to the BARA domain (residues 1–265). We define this latter construct as BECN 1-265 to clearly differentiate the nomenclature from the BARA domain construct.…”

Sensing Membrane Curvature in Macroautophagy

“…Thus the VPS34 complex includes multiple membrane interaction surfaces. How these surfaces are oriented with respect to one another could impart a second level of curvature dependence, possibly making the autophagosome-directed Beclin-containing complex significantly more dependent on local curvature 58 , as seen when comparing activity on small vesicles or GUVs 59 . In fact, the complex ordinarily exists as at least a heteropentamer (including the subunit NRBF2) 60, 61 , and the heteropentamer organizes into a dimer around a motif in NRBF2 62 , potentially allowing membrane tethering to be sensed by or driven by the VPS34 complex during autophagosome construction.…”