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

In this study we investigate the effects of glycosylation at position N99 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 glycosylation at N99 is known to play a critical, yet poorly understood role in its function. The MD 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 role in lipid redistribution across the phagophore membrane during autophagy. However, mutagenesis experiments that abolish N-glycosylation in ATG9A (ATG9A^N99Aand ATG9A^N99Dmutants) did not show a significant change in autophagy flux, suggesting that further experimental approaches, such as lipid scramblase assays, are needed to pinpoint the function of glycosylation. In this study we also observed an asymmetric protomer conformations in ATG9A, contrasting with symmetric structures obtained from 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.