Keywords:

Amino Acid Sequence, Animals, Autophagy, Autophagy-Related Protein 7, Autophagy-Related Proteins, Binding Sites, Carrier Proteins, Cell Line, Crystallography, X-Ray, Fibroblasts, Hydrophobic and Hydrophilic Interactions, Mice, Mice, Knockout, Microtubule-Associated Proteins, Models, Molecular, Molecular Sequence Data, Multienzyme Complexes, Mutation, Protein Conformation, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Protein Multimerization, Recombinant Fusion Proteins, Structure-Activity Relationship, Transfection, Ubiquitin-Conjugating Enzymes

Abstract:

<p>Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7(NTD)) recruits a unique "flexible region" from Atg3 (Atg3(FR)). The structure of an Atg7(NTD)-Atg3(FR) complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7~Atg8-Atg3)(2) complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1~UBL-E2 architecture for enzymes mediating autophagy.</p>