Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

The assembly of the autophagy initiation machinery nucleates autophagosome biogenesis, including in the PINK1- and Parkin-dependent mitophagy pathway implicated in Parkinson’s disease. The structural interaction between the sole transmembrane autophagy protein, ATG9A, and components of the ULK1 complex is one of the major missing links needed to complete a structural map of autophagy initiation. We determined the 2.4 Å x-ray crystallographic structure of the ternary structure of ATG9A C-terminal tail bound to the ATG13:ATG101 HORMA dimer, which is part of the ULK1 complex. We term the interacting portion of the extreme C-terminal part of the ATG9A tail the “HORMA dimer interacting region” (HDIR). This structure shows that the HDIR binds to the HORMA domain of ATG101 by β-sheet complementation such that the ATG9A tail resides in a deep cleft at the ATG13:ATG101 interface. Disruption of this complex in cells impairs damage induced PINK1/Parkin mitophagy mediated by the cargo receptor NDP52.

in many forms of selective autophagy, such that it is thought to be responsible for 80 bridging to the rest of the autophagy initiation machinery (Turco,Fracchiolla,and 81 Martens 2020 Here, we have identified the binding interface between ATG9A, ATG13, and ATG101. 137 We term the region of the extreme C-terminus of ATG9A that binds to the 138 ATG13:ATG101 HORMA dimer the "HORMA dimer interacting region" (HDIR). We 139 determined the structure of the human ATG9A HDIR bound to the ATG13:ATG101 140 HORMA dimer and confirmed that the interface functions in NDP52 dependent 141 mitophagy.

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To determine the affinity of the ATG9A HDIR for ATG13 HORMA :ATG101 we used 164 isothermal titration calorimetry (ITC). Serial injection of MBP-ATG9A (801-839) into a 165 cell containing ATG13 HORMA :ATG101 resulted in a Kd of 2.4 ± 0.3 mM ( Figure 1C). This 166 assay showed equal stoichiometry between the ATG13 HORMA :ATG101 dimer and 167 ATG9A 801-839 (N=0.82 ± 0.03) ( Figure 1C). This ratio is consistent with the pulldown 168 result that the trimeric assembly is not required for efficient binding. An alanine scan of 169 the ATG9A tail was performed to determine the effect of tail residue side chains on Crystal Structure of the ATG9A HDIR:ATG13 HORMA :ATG101 Ternary Complex 193 194 Having mapped the HDIR, we pursued structural analysis of the ternary complex.

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Multisequence alignment showed that the residues within the HDIR are conserved 196 across the Opisthokonta, with the exception being S. cerevisiae, whose Atg9 ortholog 197 has no sequence homology with the human HDIR ( Figure 2A). P834 and H837 are 198 highly conserved across Opisthokonta ATG9A HDIR sequences, excluding budding 199 yeasts. Alphafold2 predictive modeling (Jumper et al. 2021) was performed using the 200 primary sequence of the ATG13 HORMA , ATG101, and the ATG9A tail residues (828-839).

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To generate a set of models the primary amino acid sequences of ATG9A, 202 ATG13 HORMA , and ATG101 were submitted in varying input positions. Thirty models 203 were generated which were overlayed to determine the position of the HDIR (Supp Fig  204  2A). Two distinct positions were observed, one that involved binding only to 205 ATG13 HORMA and one which spanned both HORMA domains. The models with the HDIR 206 at the dimer interface had higher pLDDT scores. From these models, the HDIR was 207 located adjacent to the N-terminus of ATG101. We designed a fusion construct between 208 the HDIR and ATG101, using a flexible linker of 5 residues (GSDEA) to increase the 209 affinity of the ternary complex for crystallization ( Figure 2B).

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We determined the crystal structure of the ATG9A HDIR-ATG101 fusion construct 212 bound to ATG13 HORMA to 2.4 Å resolution ( Figure 2C, Table 1). Molecular replacement 213 was performed using the ATG13 HORMA :ATG101 apo structure (PDB:5C50) (Qi et al. 214 2015). The asymmetric unit contains two ATG9A HDIR: ATG13 HORMA :ATG101 215 complexes, both in a similar conformation, except that the ATG101 WF finger is in 216 different conformations in the two complexes, due to differences in the crystal packing 217 environment. ATG13 HORMA and ATG101 are in essentially the same conformation as in 218 the apo ATG13 HORMA :ATG101 structure with a root mean square deviation (RMSD) of 219 0.45 Å across all backbone atoms (Supp Figure 2B-E). Electron density at the interface 220 for ATG13 HORMA :ATG101 fit all ten residues of the HDIR, allowing confident positioning 221 of residues 830-839 ( Figure 2D). The ATG13 HORMA :ATG9A and ATG101:ATG9A 222 interfaces bury 388 Å 2 and 241 Å 2 of solvent-accessible surface area, respectively. 223 Overall, our experimental structure agrees with the Alphafold2 prediction with an RMSD 224 of 0.54 Å for backbone atoms ( Figure 2E). Our ternary crystal structure of ATG9A HDIR: 225 ATG13 HORMA :ATG101 complex determined that the HDIR binds at the interface between 226 ATG13 and ATG101 HORMA domains.

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Supplemental respectively. D) Secondary structure of ATG13 HORMA with residues that interact with 247 ATG9 HDIR shown in purple E) Secondary structure of ATG101 with residues that 248 interact with ATG9 HDIR shown in purple 249 250 251 ATG13 HORMA and ATG101 Form a Hydrophobic Groove for ATG9A HDIR Binding 252 253 The interaction of the HDIR with the ATG13 HORMA :ATG101 dimer is mediated by 254 backbone and sidechain interactions. The loop between β2' and αB in ATG13 HORMA 255 (T46-W50) is positioned to support the binding of the HDIR ( Figure 3A). This 256 rearrangement supports a hydrogen bonding interaction between ATG9A L832 and 257 Q835 and ATG13 K15 and G47 ( Figure 3A). The loop region of G43-Y45 in ATG101 258 joins a β-sheet network between β2 and HDIR residues H837-V839 ( Figure 3B). We budding yeast Atg13 to Atg9, had no effect on binding (S. W. Suzuki et al. 2015).

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Removing the WF finger of ATG101 (WF to DD) had no effect on ATG9A binding. Our  monitored to assay mitophagy. In the absence of NDP52 and ATG13, mitochondrial 300 degradation was impaired ( Figure 4A,B) as shown by the robust COXII signal after 20 301 hours of oligomycin-antimycin treatment to induce mitochondrial depolarization and 302 mitophagy. Rescue experiments with NDP52 alone were insufficient to restore COXII 303 degradation defects in ATG13 KO/penta KO cells ( Figure 4A,B). COXII degradation was 304 increased when both wild type ATG13 and NDP52 were added. Rescue experiments 305 using ATG13 W50D and Y118D mutations, which disrupt the interaction between 306 ATG13 and ATG9A in vitro ( Figure 3C), showed reduced recovery of COXII degradation 307 when compared to rescue with wild type ATG13 ( Figure 4C). These findings 308 demonstrate that the HDIR binding interface on ATG13 contributes to NDP52 309 dependent PINK1/Parkin mitophagy. Understanding the interactions responsible for autophagy initiation is central to efforts to 327 therapeutically modulate bulk and selective autophagy pathways and to understand 328 their functioning at a fundamental level. Here, we determined the structure of the 329 ATG9A HDIR bound to the ATG13:ATG101 HORMA dimer ( Figure 2C) interaction hubs regulated by conformational changes in the safety belt region (Gu,339 Desai, and Corbett 2022). This posed the question as to whether interactions of the 340 ATG13:ATG101 dimer could be regulated by conformational switching as seen for 341 Mad2 and other HORMA domains (Gu, Desai, and Corbett 2022). The interaction 342 between the ATG9A HDIR and ATG13:ATG101 is distal to and thus apparently 343 independent of the safety belt region.

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Our biochemical experiments confirmed that the ATG13 residues that bind the 346 ATG9A HDIR in the structure are required for full function. We noticed that none of 347 these mutations completely abolishes mitophagy ( Figure 4A,B certainly not the complete mechanism, nor the only mechanism. Kananngara (2021) 408 found that ULK1 could still interact with ATG9A in the absence of ATG13 as measured 409 by BioID, although this was insufficient to support normal levels of p62 autophagy. 410 Thus, at least one additional point of contact between ATG9A and the ULK1 complex is 411 likely to exist, at least in some forms of autophagy. A more significant departure from 412 this mechanism is suggested by the finding that OPTN can recruit ATG9A directly, 413 bypassing the need for the ATG9A-binding role of the ULK1 complex (Yamano et al. 414 2020). The physiological rationale for these various pathways remains largely unknown, 415 but the distinct role of different cargo receptors in neurodegeneration suggests it will be 416 important to better understand. Elucidating the molecular details of distinct initiation 417 mechanisms in various "flavors" of mitophagy should provide windows into disease 418 mechanism, and ultimately therapy. 419 420