Fig. 2: Binding sites of ATG13IDR on FIP200NTD.
From: Structure and activation of the human autophagy-initiating ULK1C:PI3KC3-C1 supercomplex
a, AlphaFold2 prediction model of the ULK1C core. b, Schematic diagram of the ULK1C core. c, Truncation and mutagenesis screening conducted by pulldown assay. Strep-Tactin resin was loaded with WT FIP200NTD–TSF to pull down various ATG13 fragments (WT 363–460, 363–398, 363–392 and 363–450 with F394D;F397D;E403K substitutions (363–450 M1)) or loaded with FIP200NTD–TSF variant (L324D;R543D;R548D) to pull down WT ATG13 fragment 363–450. The experiment was repeated four times with similar results. d,e, Close-up views of binding sites 1 and 2 of the AlphaFold2 model with the key binding residues shown. The map is contoured at 11σ. f, Strep pulldown assay. The WT FIP200NTD–TSF was used to pull down various ATG13 fragments (WT 402–517, 402–450, 450–517 and 450–517 with A452D;F453D substitutions (450–517 M2)) or FIP200NTD–TSF variant (V44D) was used to pull down WT ATG13 fragment 450–517. The results from the pulldown assays (c,f) were visualized by SDS–PAGE and Coomassie blue staining. The experiment was repeated four times with similar results. g, Close-up view of binding site 4 of the AlphaFold2 model. h, Close-up view of the ULK1MIT-binding site (site M) of ATG13MIM determined by cryo-EM. The key binding residues are shown. i,j, ATG13-KO cells stably expressing Halo–LC3B without rescue or rescued with different versions of HA–ATG13 were treated with 50 nM TMR-conjugated Halo ligand for 15 min. Following that, cells were washed with 1× PBS and treated with EBSS for the indicated time periods, harvested and analyzed by immunoblotting with indicated antibodies (i) and the percentage of the cleaved Halo band was quantified (j). Data in j are the mean ± s.d. from three independent experiments. NS, not significant. ****P < 0.0001 (two-way ANOVA). The substitution sites of each variant and their corresponding residues are shown in the insert. k, ATG13-KO cells expressing Halo–LC3B without rescue or rescued with indicated versions of HA–ATG13 were left untreated or treated with EBSS for 30 min or 45 min. Cells were harvested and analyzed by immunoblotting with the indicated antibodies. l, The ratio of phosphorylated ATG14 (p-ATG14) relative to untreated rescue WT ATG13 samples was quantified. Data in l are the mean ± s.d. from three independent experiments. ****P < 0.0001 (two-way ANOVA). The definition of mutants (bottom right) applies to i–l.
