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Updated: Jul 10, 2025
Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes
Published on: January 12, 2024
Coiled-coil-mediated dimerization of Atg16 is required for binding to the PROPPIN Atg21
Miranda Bueno-Arribas1, Celia Cruz-Cuevas1, María-Angeles Navas2
1Instituto de Investigaciones Biomédicas Sols-Morreale CSIC-UAM, Madrid, 28029, Spain.
Abstract:
PROPPINs/WIPIs are β-propeller proteins that bind phosphoinositides and contribute to the recruitment of protein complexes involved in membrane remodelling processes such as autophagosome formation and endosomal trafficking. Yeast Atg21 and mammalian WIPI2 interact with Atg16/ATG16L1 to mediate recruitment of the lipidation machinery to the autophagosomal membrane. Here, we used the reverse double two-hybrid method (RD2H) to identify residues in Atg21 and Atg16 critical for protein-protein binding. Although our results are generally consistent with the crystal structure of the Atg21-Atg16 complex reported previously, they also reveal that dimerization of the Atg16 coiled-coil domain is required for Atg21 binding. Furthermore, most of the residues identified in Atg21 are conserved in WIPI2 and we showed that these residues also mediate ATG16L1 binding. Strikingly, these residues occupy the same position in the β-propeller structure as residues in PROPPINs/WIPIs Hsv2 and WIPI4 that mediate Atg2/ATG2A binding, supporting the idea that these proteins use different amino acids at the same position to interact with different autophagic proteins. Finally, our findings demonstrate the effectiveness of the RD2H system to identify critical residues for protein-protein interactions and the utility of this method to generate combinatory mutants with a complete loss of binding capacity.
Insights
Yeast Atg21 and mammalian WIPI2 proteins bind Atg16/ATG16L1, crucial for autophagosome formation. This study identified key residues in Atg21 and Atg16 essential for this interaction using the RD2H system.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- PROPPINs/WIPIs are β-propeller proteins involved in membrane remodeling, including autophagosome formation and endosomal trafficking.
- Yeast Atg21 and mammalian WIPI2 interact with Atg16/ATG16L1 to recruit lipidation machinery to the autophagosomal membrane.
Purpose of the Study:
- To identify critical residues in Atg21 and Atg16 essential for their protein-protein binding using the reverse double two-hybrid (RD2H) method.
- To investigate the role of Atg16 coiled-coil domain dimerization in Atg21 binding.
- To assess the conservation of identified residues in WIPI2 and their role in ATG16L1 binding.
Main Methods:
- Reverse double two-hybrid (RD2H) system to identify critical residues for protein-protein interactions.
- Analysis of protein complex crystal structures.
- Site-directed mutagenesis to create combinatory mutants.
Main Results:
- Dimerization of the Atg16 coiled-coil domain is necessary for Atg21 binding.
- Identified conserved residues in Atg21 that mediate ATG16L1 binding, occupying similar structural positions as residues involved in other autophagic protein interactions.
- Demonstrated the effectiveness of the RD2H system in identifying interaction hotspots and generating mutants with complete loss of binding.
Conclusions:
- The RD2H system is a powerful tool for dissecting protein-protein interactions in autophagy.
- Specific residues in Atg21 and WIPI2 are critical for binding Atg16/ATG16L1, highlighting conserved interaction mechanisms in autophagy.
- Structural insights suggest convergent evolution in protein-protein interaction interfaces within the autophagy machinery.
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