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Structural basis for membrane recruitment of ATG16L1 by WIPI2 in autophagy

  1. Lisa M Strong
  2. Chunmei Chang
  3. Julia F Riley
  4. C Alexander Boecker
  5. Thomas G Flower
  6. Cosmo Z Buffalo
  7. Xuefeng Ren
  8. Andrea KH Stavoe
  9. Erika LF Holzbaur
  10. James H Hurley  Is a corresponding author
  1. Department of Molecular and Cell Biology, University of California, Berkeley, United States
  2. California Institute for Quantitative Biosciences, University of California, Berkeley, United States
  3. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, United States
  4. Department of Physiology, University of Pennsylvania Perelman School of Medicine, United States
  5. Department of Neurobiology and Anatomy, The University of Texas Health Science Center at Houston McGovern Medical School, United States
Research Article
Cite this article as: eLife 2021;10:e70372 doi: 10.7554/eLife.70372
9 figures, 1 table and 3 additional files

Figures

WIPI2d:ATG16L1 W2IR structure.

Structure of WIPI2d bound to ATG16L1 W2IR. (A) Annotated WIPI2d and ATG16L1 domain schematics. WIPI2d construct for crystallography is shown and W2IR from ATG16L1. (B, C) The ribbon diagram of the WIPI2d complex with ATG16L1 W2IR from the (B) bottom and (C) side views. Each blade is colored in accordance with (A). (D) Composite omit map of ATG16L1 W2IR. Modeled ATG16L1 is shown as red carton and the composite omit 2mFo-DFc map contoured at 1σ is shown in gray.

Interactions at the interface.

Analysis of the Interface. (A) Overall electrostatic surface and (B) closer view of electrostatic surface with ATG16 W2IR shown as a cartoon and key residues labeled. (C) Overall hydrophobic surface of WIPI2d and (D) closer view of the hydrophobic interface with key residues labeled where yellow represents hydrophobic regions. (E) A cartoon and stick representation of hydrogen bonds between ATG16 and WIPI2d shown as black dotted lines with distances noted and key residues shown as sticks.

WIPI2d Interfacial mutants decrease ATG16L1 binding.

Key interacting residues shown as sticks in cartoon representation of WIPI2d:ATG16L1 interface shown from (A) the WIPI2d face or (B) down the ATG16L1 helix. (C) Inputs for the (D) Pull-down assays of mutant WIPI2d constructs and wild type with GST-ATG16L1 W2IR. GSH resin was used to pull-down GST-ATG16L1 W2IR from purified protein mixture. The pull-down results were performed in triplicates and visualized by SDS–PAGE and Coomassie blue staining.

Figure 3—source data 1

Uncropped SDS-PAGE gels for Figure 3.

Uncropped gel used in Figure 3C,D with lanes labeled similarly.

https://cdn.elifesciences.org/articles/70372/elife-70372-fig3-data1-v2.zip
WIPI2d mutants disrupt E3 recruitment and LC3 lipidation on GUVs.

(A) The schematic drawing illustrates the reaction setting. Colors indicate fluorescent protein-fused components. Components in gray are not labeled but are present in the reaction. (B) Representative confocal images of GUVs showing E3 membrane binding and LC3B lipidation. PI3KC3-C1, WIPI2d WT or mutant, E3-GFP, ATG7, ATG3, mCherry-LC3B, and ATP/Mn2+ were incubated with GUVs (64.8% DOPC: 20% DOPE: 5% DOPS: 10% POPI: 0.2% Atto647 DOPE) at room temperature. Images taken at 30 min were shown. Scale bars, 10 µm. (C) Quantification of relative intensities of E3-GFP and mCherry-LC3B on GUV membranes in (A) (means ± SDs are shown; N = 40). p≥0.5: (ns); 0.01<p<0.05: (*); 0.001<p<0.01: (**); p<0.001 (***); p<0.0001 (****).

Altering the electrostatic interface of WIPI2 impairs starvation-induced autophagy in MEFs.

(A) Representative maximum projections of LC3 staining and either Halo or Halo-WIPI2 signal in WT or WIPI2 knockout (KO) cells (indicated on left) following 2 hr of starvation and 100 nM BafA treatment. Scale bar 15 µm. (B) Number of LC3-positive autophagosomes in either WT cells transfected with Halo, WIPI2 KO cells transfected with Halo, or WIPI2 KO cells transfected with the indicated Halo-tagged WIPI2 construct (labeled by mutation). (C) Number of discrete WIPI2 puncta under the conditions described in (B), measured using maximum projections of Halo-tag fluorescence. Experimental replicates are color-coded, with translucent dots representing individual measurements from each replicate and opaque dots, the corresponding arithmetic mean of that replicate. Error bars ± SEM; n = 4 independent experiments; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 based on a one-way ANOVA with Tukey’s multiple comparisons between all conditions.

Reconstitution of membrane recruitment of WIPI2d mutants.

(A) Representative confocal images of GUVs showing membrane binding of mCherry-WIPI2d. PI3KC3-C1, mCherry-WIPI2d WT or mutant, E3-GFP were incubated with GUVs (64.8% DOPC: 20% DOPE: 5% DOPS: 10% POPI: 0.2% Atto647 DOPE) at room temperature. Images taken at 30 min were shown. Scale bars, 10 µm. (B) Quantification of relative intensities of mCherry-WIPI2d on GUV membranes in (A) membranes (means ± SDs are shown; N = 40). (C) Quantification of confocal images of GUVs (69.8% DOPC: 20% DOPE: 5% DOPS: 5% DOPI(3)P: 0.2% Atto647 DOPE) showing membrane binding of mCherry-WIPI2d. mCherry-WIPI2d WT or mutant were incubated with GUVs at room temperature for 30 min and then imaged. (Means ± SDs are shown; N = 40). p≥0.5: (ns); 0.01<p<0.05: (*); 0.001<p<0.01: (**); p<0.001 (***); p<0.0001 (****).

Comparing WIPI2d and WIPI3 structures and binding modes.

Comparison of WIPI2d and WIPI3. Alignment of WIPI2d and WIPI3 (A) structure and (B) sequence based on structures with W2IR residues denoted with white squares, W34IR with black, and from both with gray. Electrostatic surface comparison of (C) WIPI2d and (D) WIPI3.

Comparison of WIPI2 and Atg21 binding to Atg16.

(A) Structural alignment of WIPI2 (PDB: 7MU2) and Atg21 (PDB: 6RGO; indigo) structures bound to Atg16. (B) Sequence alignment of Atg16 ß-propeller binding residues based on structure. Residues for WIPI2 and Atg21 binding are in highlighted in red and purple, respectively. (C) Electrostatic potential of Atg21 with overlay of AgAtg16 and ATG16L1 in purple and red, respectively. Key interacting residues are shown in sticks and labeled. Model of ATG12–5-16 performing LC3 lipidation on the autophagic membrane with (D) WIPI2 recruitment in humans with Helix one membrane binding is labeled (Lystad et al., 2019), and a secondary upward conformation is shown in faded colors versus (E) Atg21 recruitment in yeast.

WIPI1-4 comparison.

Comparison of electrostatic surface potential of (A) WIPI1-4. (B) Hydrophobic surface of WIPI1 with predicted ATG16L1 W2IR shown as cartoon. (C, D) Alignment of WIPI2d crystal structure and WIPI1 homology structure with WIPI1 shown as light green and key residues labeled in the same color as structure.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)HeLa human epithelial cell lineATCCCCL-2Authenticated by STR profiling; tested negative for mycoplasma
Cell line (Homo sapiens)WIPI2-KO cells: HeLa cell line gene-edited to knockout WIPI2 expressionFischer et al., 2020
Recombinant DNA reagentpHTC HaloTagPromegaG7711
Recombinant DNA reagentHalo-WIPI2-WT (Homo sapiens) plasmid for transfectionStavoe et al., 2019175,025Available from Addgene
Recombinant DNA reagentHalo-WIPI2-H85E (Homo sapiens) plasmid for transfectionModified from Halo-WIPI2-WT in Stavoe et al., 2019175,027Available from Addgene
Recombinant DNA reagentHalo-WIPI2-K88E (Homo sapiens) plasmid for transfectionModified from Halo-WIPI2-WT in Stavoe et al., 2019175,028Available from Addgene
Recombinant DNA reagentHalo-WIPI2-I92E(Homo sapiens) plasmid for transfectionModified from Halo-WIPI2-WT in Stavoe et al., 2019175,029Available from Addgene
Recombinant DNA reagentHalo-WIPI2-R108E (Homo sapiens) plasmid for transfectionStavoe et al., 2019176,004Available from Addgene
Recombinant DNA reagentHalo-WIPI2-H85/K88/I92E (Homo sapiens) plasmid for transfectionModified from Halo-WIPI2-WT in Stavoe et al., 2019175,033Available from Addgene
AntibodyAnti-LC3B, (Rabbit polyclonal) primary antibodyAbcamCat.#ab48394IF (1:1000)
AntibodyAnti-Rabbit AlexaFluor488, (Goat polyclonal) secondary antibodyThermoFisherCat.#A11034IF (1:1000)
Chemical compound, drugTMRDirect Halo LigandPromegaCat.#G299137.5 nM final concentration
Software, algorithmFIJIPMID:22743772
Software, algorithmIlastikPMID:31570887
Software, algorithmAdobe Illustrator 2021Adobe
Software, algorithmPrism 9GraphPad
Other35 mm #1.5 glass bottom imaging dishesMatTekCat.# P35G-1.5–20 C
OtherEBSSThermoFisherCat.# 24010043
Cell line (Homo sapiens)HEK GnTiATCCCRL-3022
Recombinant DNA reagentpCAG-WIPI2d-cs-TEVFracchiolla et al., 2020
Recombinant DNA reagentpCAG-WIPI2d10-364Δ263–295-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dH85E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dK88E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dI92E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dC93E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dR108E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dR125E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dK128E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2d-cs-TEVFracchiolla et al., 2020
Recombinant DNA reagentpCAG-mcherry-WIPI2dH85E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dK88E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dI92E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dC93E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dR108E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dR125E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dK128E-cs-TEVThis paperMaterials and methods section: Plasmids
Recombinant DNA reagentpLEXm-GST-TEV-ATG14RRID:Addgene_99,329
Recombinant DNA reagentpCAG-TSF-TEV-BECN1RRID:Addgene_99,328
Recombinant DNA reagentpCAG-TSF-TEV-VPS34RRID:Addgene_99,327
Recombinant DNA reagentpCAG-VPS35Stjepanovic et al., 2017
Recombinant DNA reagentpGBdest-ATG12-10xHis-TEV-ATG5-10xHis-TEVcs-ATG16L1-GFP-TEVcs-StrepII, ATG7, ATG10Fracchiolla et al., 2020
Recombinant DNA reagentpFast BacHT(B)–6xHis-TEV-ATG7Fracchiolla et al., 2020
Recombinant DNA reagentpET Duet-1-6xHis-TEV-ATG3Fracchiolla et al., 2020
Recombinant DNA reagentpET Duet-1-6xHis-TEV-mCherry-LC3B-Gly(∆5 C)Zaffagnini et al., 2018
Other96–2 well,INTELLI-PLATE (original) trayMolecular Dimensions, Maumee, OH
OtherGreiner pre-greased 24 well Combo Plate (SBS format) with lidMolecular Dimensions, Maumee, OH
Software, algorithmNikon Elements microscope imaging software 4.60Nikon Corporation, Tokyo, Japanhttps://www.nikoninstruments.com/Products/Software/NIS-Elements-Advanced-Research/NIS-Elements-Viewer
OtherGlutathione Sepharose 4B GST-tagged protein purification resinGE healthcare, Chicago, ILCat.# 17075605
OtherStrep-Tactin Superflow high capacity 50 % suspensionIBA Lifesciences,Göttingen, GermanyCat.# 2-1208-010
Software, algorithmphenix.refinePMID:20124702,
22505256, 31588918
RRID:SCR_016736
Software, algorithmXDSPMID:20124692RRID:SCR_015652
Software, algorithmPOINTLESSPMID:21460446RRID:SCR_014218
Software, algorithmPHASERPMID:19461840RRID:SCR_014219
Software, algorithmPyMolPyMol (pymol.org)RRID:SCR_000305
Software, algorithmAPBSPMID:11517324RRID:SCR_008387
Software, algorithmSWISS-MODELPMID:29788355RRID:SCR_018123
Software, algorithmCootPMID:20383002RRID:SCR_014222

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