Abstract
WIPI proteins (WIPI1-4) are mammalian PROPPIN family phosphoinositide effectors essential for autophagosome biogenesis. In addition to phosphoinositides, WIPI proteins can recognize a linear WIPI-interacting-region (WIR)-motif, but the underlying mechanism is poorly understood. Here, we determine the structure of WIPI3 in complex with the WIR-peptide from ATG2A. Unexpectedly, the WIR-peptide entwines around the WIPI3 seven-bladed β-propeller and binds to three sites in blades 1–3. The N-terminal part of the WIR-peptide forms a short strand that augments the periphery of blade 2, the middle segment anchors into an inter-blade hydrophobic pocket between blades 2–3, and the C-terminal aromatic tail wedges into another tailored pocket between blades 1–2. Mutations in three peptide-binding sites disrupt the interactions between WIPI3/4 and ATG2A and impair the ATG2A-mediated autophagic process. Thus, WIPI proteins recognize the WIR-motif by multi-sites in multi-blades and this multi-site-mediated peptide-recognition mechanism could be applicable to other PROPPIN proteins.
Highlights
WIPI proteins (WIPI1-4) are mammalian PROPPIN family phosphoinositide effectors essential for autophagosome biogenesis
To investigate the mechanism underlying the recognition of the linear WIR-motif by WIPI proteins, we initiated this work with characterizing the interactions between WIPI proteins and the WIPIbinding fragments of ATG2A
Both WIPI3 and WIPI4 were found to interact with ATG2A20, WIPI3 exhibited excellent protein sample quality and was chosen for biochemical and structural studies[25]
Summary
WIPI-interacting region in ATG2A for binding to WIPI3. To investigate the mechanism underlying the recognition of the linear WIR-motif by WIPI proteins, we initiated this work with characterizing the interactions between WIPI proteins and the WIPIbinding fragments of ATG2A. The C-terminus of the WIR-peptide from one molecule is linked to the N-terminus of WIPI3-Δloop from another molecule (Supplementary Fig. 2), which accidentally promotes the packing of neighboring molecules for crystallization This may explain why WIPI3-Δloop only with N-terminal fusion of the WIRpeptide could be crystallized. Based on the crystal packing analysis, the fusion of the WIR-peptide to WIPI3-Δloop did not induce any distortion of the packing between them and had little impact on the complex structure (Supplementary Fig. 2). The two deleted loops in WIPI3 are far away from these inter-blade sites and would not impact on the complex formation, consistent with the similar binding affinities of WIR-peptide for WIPI3 and WIPI3-Δloop WIPI3 contains two conserved phosphoinositide-binding sites in blades 5–6 (characterized with the signature “LRRG” motif), which do not overlap but are
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