Insights into autophagosome maturation revealed by the structures of ATG5 with its interacting partners

Autophagy is a bulky catabolic process that responds to nutrient homeostasis and extracellular stress signals and is a conserved mechanism in all eukaryotes. When Autophagy is induced, cellular components are sequestered within an autophagosome and finally degraded by subsequent fusion with a lysosome. During this process, the ATG12-ATG5 conjugate requires 2 different binding partners, ATG16L1 for autophagosome elongation and TECPR1 for lysosomal fusion. In our current study, we describe the crystal structures of human ATG5 in complex with an N-terminal domain of ATG16L1 as well as an internal AIR domain of TECPR1. Both binding partners exhibit a similar α-helical structure containing a conserved binding motif termed AFIM. Furthermore, we characterize the critical role of the C-terminal unstructured region of the AIR domain of TECPR1. These findings are further confirmed by biochemical and cell biological analyses. These results provide new insights into the molecular details of the autophagosome maturation process, from its elongation to its fusion with a lysosome.

AFIM, ATG5 (5)-interacting motif; AIR, ATG12–ATG5-interacting region; ATG, autophagy-related; ATG12; ATG16; ATG16N69, ATG16L1 N-terminal 69 residues; ATG5; DAPI, 4’, 6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; FLuc, firefly luciferase; FP, fluorescent polarization; GAL4-BD, GAL4-DNA binding domain; GFP, green fluorescent protein; HR, helix rich; ITC, isothermal titration calorimetry; MR, molecular replacement; PE, phosphatidylethanolamine; PH, pleckstrin homology; PtdIns3P, phosphatidylinositol 3-phosphate; RLuc, Renilla luciferase; SPR, surface plasmon resonance; TECAIR, TECPR1 AIR; TECPR1; TECPR1, tectonin β-propeller repeat containing 1; UFD, ubiquitin-fold domain; Ubl, ubiquitin-like protein; VP16-AD, herpes simplex virus VP16 transcription activation domain; autophagy; buffer A, buffer containing 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 1 mM TCEP; crystal structure; lysosome fusion; r.m.s., root-mean-square.