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

  • Curated by eLife

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    Evaluation Summary:

    This paper describes the crystal structure of two key components of the autophagy system, the PI3P-binding protein WIPI2d in complex with its interaction region in the hATG8 E3 ligase scaffold component ATG16L1. The paper provides interesting new data and demonstrates the requirements for association of WIPI2d with membranes. Functional studies in cells provide evidence that mutation of residues at the interface for ATG16L1 binding affects function, although additional studies would support loss of function versus a dominant negative effect.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12–5–16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12–5–16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207–230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12–5–16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12–5–16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other.

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  1. Version published to 10.7554/elife.70372 on eLife
  2. eLife

    Evaluation Summary:

    This paper describes the crystal structure of two key components of the autophagy system, the PI3P-binding protein WIPI2d in complex with its interaction region in the hATG8 E3 ligase scaffold component ATG16L1. The paper provides interesting new data and demonstrates the requirements for association of WIPI2d with membranes. Functional studies in cells provide evidence that mutation of residues at the interface for ATG16L1 binding affects function, although additional studies would support loss of function versus a dominant negative effect.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    This paper describes the structure of WIPI2d in association with a region of ATG16L1 that interacts with WIPI. Previous work has demonstrated that WIPI proteins bind to PI3P on the surface of phagophores to initiate autophagy. WIPI also associates with ATG16 orthologs to template assembly of the phagophore expansion machinery. The authors map the interface, and also perform biochemical and cell biological experiments that support the molecular structure.

    Analysis of mutations within the interaction interface reveal the residues required for interaction, confirming the structural data. This information will be very important in efforts to biochemically reconstitute the initial stages of autophagy. This work also reveals the complications of examining mutations in complex interaction surfaces in cells, particularly in cases where multivalent interactions drive the process. The results also explain why there are multiple WIPI proteins in the human genome, reflecting distinct mechanisms of recruitment of ATG16 and ATG2 orthologs to the appropriate membrane.

  4. eLife

    Reviewer #2 (Public Review):

    This manuscript from Hurley and colleagues uses x-ray crystallography to determine the structure of WIPI-2d in complex with a peptide representing the WIPI-2 binding domain of ATG16L1. WIPI-2 proteins play a crucial role early in autophagosome biogenesis by binding phosphoinositides and then recruiting the ATG16L1 complex to the growing autophagosome membrane. WIPI-2 is a member of the mammalian PROPPIN family of WD-repeat proteins which includes WIPI-1, WIPI-2, WIPI-3 and WIPI-4, but until now, only the WIPI-3 structure had been determined. As expected, the authors reveal that WIPI-2d adopts the classic 7-bladed WD-repeat structure. Further they show that the ATG16L1 helix previously described as a WIPI-2 binding site (Dooley/Tooze 2014), interacts specifically between the 2nd and 3rd blade of the WD-repeat structure through combinations of hydrophobic and charged amino acids on the amphipathic helix. From this result they were able to map the crucial amino acids involved in protein-protein interaction, many of which had previously been identified through elegant biochemistry/mutational analysis experiments (in Dooley..Tooze, 2014), further confirming the validity of the authors' structure.

    They confirm that the crystallographically-determined amino acids forming the peptide binding site are needed for peptide recruitment in an affinity pull-down experiment and then further show that these same amino acids are needed to support recruitment of the full ATG16L1 complex to membranes in an elegant in vitro reconstitution assay of LC3-lipid attachment. Intriguingly, they also observe that recruitment of WIPI2 itself to membranes in the presence of ATG16L1, is reduced when mutations are introduced into the ATG16L1 binding site. It is not clear from their experiments whether this reduction in membrane binding reflects interactions with ATG16L1 or is a general impairment to WIPI2-phosphoinositide interaction. Experiments testing the membrane binding of WIPI2 alone following mutagenesis would help tease out these differences.

    The authors also examine whether mutation of the ATG16L1 binding site they describe impacts autophagy events in cells. To do this, they use siRNA knockdown of WIPI2 and then re-express either wildtype or mutant forms of WIPI2 and follow both WiPI2 puncta formation (suggesting WIPI2 recruitment to new membranes) and LC3 puncta formation (indicative of autophagosome formation), very similar to the original assay design of Dooley/Tooze. The authors observe a modest reduction on LC3 numbers for essentially all mutants tested, thus the direction of the experiment is consistent with their hypothesis. However, the very mild impact here of mutating these presumably key residues is surprising given the all-or-nothing behavior of some mutants in vitro. This assay is essentially a test for dominant-negative behavior and will always be sensitive to the level of knockdown achieved as the expressed protein is competing against the wildtype reserves. The authors do not show what the impact of the knockdown alone is on their system and one possibility is that that the remaining WiPI2 observable by western blot is largely sufficient to support autophagy. The authors also note that the impacts of mutation in cells does not follow the same rank order of impacts in vitro. A more dramatic starting knockdown or a full knockout experiment followed by rescue with wildtype or mutants would likely provide a clearer signal-to-noise in interpreting the significance of these amino acids in supporting ATG16L1 activities in cells.

  5. eLife

    Reviewer #3 (Public Review):

    In this manuscript, Strong et al. sought to address how WIPI2 recruits the LC3 E3 ligase scaffold component ATG16L1 to the nascent autophagosomal membrane. For this purpose, the authors determined a high-resolution structure of a consensus WIPI2 bound to the WIPI2-interacting region (W2IR) of ATG16L1 using x-ray crystallography. Their model WIPI2 adopted a 7-bladed beta propeller to which the W2IR alpha helix bound in a hydrophobic groove situated between two blades (#2 and #3). Importantly, mutational analysis of W2IR interface residues in WIPI2 confirmed their critical roles in GST pulldown and LC3 lipidation reconstitution experiments in vitro as well as LC3 and WIPI puncta formation assays in starved cells. Rounding off, the authors compared their WIPI2-ATG16L1 W2IR structure to a recently solved structure of WIPI3, which was shown to specifically bind to ATG2A and generated structural models for the two remaining WIPI family members, namely WIPI1 and WIPI4. Overall, Strong and colleagues provide compelling mechanistic insights into WIPI2's role in mediating E3 recruitment and LC3 lipidation. Moreover, the authors' structural data of WIPI2 will help to rationalize functions of the other WIPI proteins.

  6. Version published to 10.1101/2021.05.14.444175v1 on bioRxiv