title: Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy.
abstract: Autophagy, an essential eukaryotic homeostasis pathway, allows the sequestration of unwanted, damaged, or harmful cytoplasmic components in vesicles called autophagosomes, permitting 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 antiparallel, 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 heterodimerization, allowing us to identify ATG14 interface residues. Finally, we verify the role of BECN1 and ATG14 interface residues in binding by assessing the impact of point mutations of these residues on co-immunoprecipitation of the partner and demonstrate that these mutations abrogate starvation-induced upregulation 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 that are important for BECN1:ATG14 heterodimerization and for autophagy.
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University , Fargo, North Dakota 58108-6050, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Mei
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University , Fargo, North Dakota 58108-6050, United States.
authors.forename: Minfei
authors.initials: M
authors.lastname: Su
authors.affiliation: GMCA@APS, X-ray Science Division, Advanced Photon Source, Argonne National Laboratory , Argonne, Illinois 60439, United States.
authors.forename: Ruslan
authors.initials: R
authors.lastname: Sanishvili
authors.affiliation: Bio-CAT, Advanced Photon Source , 9700 South Cass Avenue, Building 435B, Argonne, Illinois 60439, United States.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Chakravarthy
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University , Fargo, North Dakota 58108-6050, United States.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Colbert
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University , Fargo, North Dakota 58108-6050, United States.
authors.forename: Sangita C
authors.initials: SC
authors.lastname: Sinha
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D001343:Autophagy / Q000502:physiology*; D000071183:Autophagy-Related Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D000071186:Beclin-1 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D013217:Starvation / Q000503:physiopathology; D014961:X-Ray Diffraction
chemical_list: C539000:ATG14 protein, human; D033942:Adaptor Proteins, Vesicular Transport; D000071183:Autophagy-Related Proteins; C491997:BECN1 protein, human; D000071186:Beclin-1; D011993:Recombinant Fusion Proteins