ATG16L1 is equipped with two distinct WIPI2-binding sites to drive autophagy.

The recruitment of ATG12-ATG5-ATG16L1 complex to phagophore mediated by the specific interaction between ATG16L1 and WIPI2, is pivotal to the formation of autophagosomes during macroautophagy. Recently, we reported that ATG16L1 contains two distinct WIPI2-binding sites, the previously reported WIPI2-binding site (WBS1), and the newly identified site (WBS2). By determining the crystal structures of WIPI2 with ATG16L1 WBS1 and WBS2 respectively, we uncovered that, unlike ATG16L1 WBS1, ATG16L1 WBS2 and its binding mechanism to WIPI2 are conserved from yeast to mammals. Using cell-based functional assays, we further demonstrated that the integrity of two WIPI2-binding sites of ATG16L1 is essential for normal autophagic flux. In summary, our study provided mechanistic insights into the interaction of two key autophagic proteins, ATG16L1 and WIPI2, and revealed a dual-binding-site mode adopted by ATG16L1 to associate with WIPI2.Abbreviations: ATG: autophagy-related protein; CCD: coiled-coil domain; ITC: isothermal titration calorimetry; PI3KC3-C1: class III phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; ULK: Unc-51-like kinase; WBS: WIPI2-binding site; WIPI: WD repeat domain phosphoinositide-interacting protein.