Human ATG3 contains a non-canonical LIR motif crucial for its enzymatic activity in autophagy

Farnung, Jakob; Muhar, Matthias; Liang, Jin Rui; Tolmachova, Kateryna A.; Benoit, Roger; Corn, Jacob E.; Bode, Jeffrey W.

doi:10.1101/2022.08.02.502437

Cited by 3 publications

(3 citation statements)

References 48 publications

(60 reference statements)

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“…Overexpression of the ATG3 AIM peptide reduced the formation of ATG8CL puncta ( Fig 3G ), indicating that it can block autophagy when overproduced in plant cells. In agreement with our findings, a recent report showed human ATG3 interacts with LC3 through the noncanonical LIR sequence WXXT [ 50 ]. These results show that the AF2-multimer can predict both canonical/noncanonical AIM/LIRs and can serve as a powerful tool when combined with the molecular phylogeny analysis to provide new autophagy understanding.…”

Section: Resultssupporting

confidence: 94%

AlphaFold2-multimer guided high-accuracy prediction of typical and atypical ATG8-binding motifs

et al. 2023

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Macroautophagy/autophagy is an intracellular degradation process central to cellular homeostasis and defense against pathogens in eukaryotic cells. Regulation of autophagy relies on hierarchical binding of autophagy cargo receptors and adaptors to ATG8/LC3 protein family members. Interactions with ATG8/LC3 are typically facilitated by a conserved, short linear sequence, referred to as the ATG8/LC3 interacting motif/region (AIM/LIR), present in autophagy adaptors and receptors as well as pathogen virulence factors targeting host autophagy machinery. Since the canonical AIM/LIR sequence can be found in many proteins, identifying functional AIM/LIR motifs has proven challenging. Here, we show that protein modelling using Alphafold-Multimer (AF2-multimer) identifies both canonical and atypical AIM/LIR motifs with a high level of accuracy. AF2-multimer can be modified to detect additional functional AIM/LIR motifs by using protein sequences with mutations in primary AIM/LIR residues. By combining protein modelling data from AF2-multimer with phylogenetic analysis of protein sequences and protein–protein interaction assays, we demonstrate that AF2-multimer predicts the physiologically relevant AIM motif in the ATG8-interacting protein 2 (ATI-2) as well as the previously uncharacterized noncanonical AIM motif in ATG3 from potato (Solanum tuberosum). AF2-multimer also identified the AIM/LIR motifs in pathogen-encoded virulence factors that target ATG8 members in their plant and human hosts, revealing that cross-kingdom ATG8-LIR/AIM associations can also be predicted by AF2-multimer. We conclude that the AF2-guided discovery of autophagy adaptors/receptors will substantially accelerate our understanding of the molecular basis of autophagy in all biological kingdoms.

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Section: Resultssupporting

confidence: 94%

AlphaFold2-multimer guided high-accuracy prediction of typical and atypical ATG8-binding motifs

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Overexpression of the ATG3 AIM peptide reduced the formation of ATG8CL puncta (Figure 3G), indicating that it can block autophagy when overproduced in plant cells. In agreement with our findings a recent report showed human ATG3 interacts with LC3 through the non-canonical LIR sequence WXXT [45]. These results show that the AF2-multimer can predict both canonical/non-canonical AIM/LIRs and can serve as a powerful tool when combined with the molecular phylogeny analysis to provide new autophagy understanding.…”

Section: Resultssupporting

confidence: 93%

AF2-multimer guided high accuracy prediction of typical and atypical ATG8 binding motifs

Ibrahim

Khandare

Mirkin

et al. 2022

Preprint

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Macroautophagy/autophagy is an intracellular degradation process central to cellular homeostasis and defense against pathogens in eukaryotic cells. Regulation of autophagy relies on hierarchical binding of autophagy cargo receptors and adaptors to ATG8/LC3 protein family members. Interactions with ATG8/LC3 are typically facilitated by a conserved, short linear sequence, referred to as the ATG8/LC3 interacting motif/region (AIM/LIR), present in autophagy adaptors and receptors as well as pathogen virulence factors targeting host autophagy machinery. Since the canonical AIM/LIR sequence can be found in many proteins, identifying functional AIM/LIR motifs has proven challenging. Here we show that protein modelling using Alphafold-Multimer (AF2-multimer) identifies both canonical and atypical AIM/LIR motifs with a high level of accuracy. AF2-multimer can be modified to detect additional functional AIM/LIR motifs by using protein sequences with mutations in primary AIM/LIR residues. By combining protein modelling data from AF2-multimer with phylogenetic analysis of protein sequences and protein-protein interaction assays, we demonstrate that AF2- multimer predicts the physiologically relevant AIM motif in the ATG8-interacting protein 2 (ATI-2) as well as the previously uncharacterized non-canonical AIM motif in ATG3 from potato (Solanum tuberosum). AF2-multimer also identified the AIM/LIR motifs in pathogen-encoded virulence factors that target ATG8 members in their plant and human hosts, revealing that cross-kingdom ATG8- LIR/AIM associations can also be predicted by AF2-multimer. We conclude that the AF2-guided discovery of autophagy adaptors/receptors will substantially accelerate our understanding of the molecular basis of autophagy in all biological kingdoms.

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“…ATG3’s N-terminal was still folded as an α -helix in these models, connecting via a short linker to the body part of ATG3 containing a large, disordered region. LC3 formed a complex with ATG3 possibly via the LC3 interacting region (LIR) of ATG3 ( 40 ), and we additionally modeled the thioester bond between CYS264 of ATG3 and GLY120 of LC3. We inserted the Alpha Fold model into a solvated lipid bilayer and ran long equilibrium MD simulations.…”

Section: Resultsmentioning

confidence: 99%

Unique Amphipathicα-helix Drives Membrane Insertion and Enzymatic Activity of ATG3

Nishimura

Lazzeri

Mizushima

et al. 2023

Preprint

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Autophagosome biogenesis requires a localized perturbation of lipid membrane dynamics and a unique protein-lipid conjugate. Autophagy-related (ATG) proteins catalyze this biogenesis on cellular membranes, but the underlying molecular mechanism remains unclear. Focusing on the final step of the protein-lipid conjugation reaction, ATG8/LC3 lipidation, we show how membrane association of the conjugation machinery is organized and fine-tuned at the atomistic level. Amphipathic α-helices in ATG3 proteins (AHATG3) are found to have low hydrophobicity and to be less bulky. Molecular dynamics simulations reveal that AHATG3regulates the dynamics and accessibility of the thioester bond of the ATG3~LC3 conjugate to lipids, allowing covalent lipidation of LC3. Live cell imaging shows that the transient membrane association of ATG3 with autophagic membranes is governed by the less bulky-hydrophobic feature of AHATG3. Collectively, the unique properties of AHATG3facilitate protein-lipid bilayer association leading to the remodeling of the lipid bilayer required for the formation of autophagosomes.

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Human ATG3 contains a non-canonical LIR motif crucial for its enzymatic activity in autophagy

Cited by 3 publications

References 48 publications

AlphaFold2-multimer guided high-accuracy prediction of typical and atypical ATG8-binding motifs

AlphaFold2-multimer guided high-accuracy prediction of typical and atypical ATG8-binding motifs

AF2-multimer guided high accuracy prediction of typical and atypical ATG8 binding motifs

Unique Amphipathicα-helix Drives Membrane Insertion and Enzymatic Activity of ATG3

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