Role of N -glycosylation as a determinant of ATG9A conformations and activity

This study investigates the effects of N99 glycosylation on the structural dynamics and lipid scrambling activity of ATG9A, a key autophagy protein, using microsecond all-atom molecular dynamics (MD) simulations. ATG9A is an integral membrane protein involved in autophagosome biogenesis, and its N-glycosylation at N99 plays a critical, yet poorly understood role in its function. The simulations revealed that the hydrophilic central cavity of ATG9A supports lipid reorientation and partial transbilayer movements, consistent with its lipid scrambling activity observed experimentally. N-glycosylation at N99 was found to enhance cooperative interactions between protomers, facilitating lipid insertion and traversal within the central cavity. These findings align with the proposed mechanism of ATG9A’s role in lipid redistribution across the phagophore membrane during autophagy. However, mutagenesis experiments that abolished N-glycosylation in ATG9A (ATG9A ^N99A and ATG9A ^N99D mutants) did not show a marked change in autophagy flux, suggesting that further experimental approaches, such as lipid scramblase assays, may be needed to more clearly define the glycosylation’s impact. The study also observed asymmetric protomer conformations in ATG9A, contrasting with symmetric structures seen in cryo-EM, suggesting that the structural heterogeneity of the protein could be further explored in cryo-EM datasets. Overall, the study highlights the importance of incorporating glycosylation in computational studies of membrane proteins and offers valuable insights into the molecular mechanisms of lipid transport in autophagy, with potential implications for other lipid scramblases and flippases.