Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

doi:10.21203/rs.3.rs-5058991/v1

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Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

https://doi.org/10.21203/rs.3.rs-5058991/v1

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Macroautophagy maintains cellular and organismal homeostasis, and entails de novo synthesis of double-membrane autophagosome. The effective formation of autophagosome requires the recruitment of the ATG12~ATG5-ATG16L1complex to the pre-autophagosomal structure by relevant ATG16L1-binding autophagic factors including FIP200. However, the molecular mechanism governing the specific interaction of ATG16L1 with FIP200 remains elusive. Here, we uncover that ATG16L1 contains a FIP200-interacting region (FIR), which not only can directly bind to the Claw domain of FIP200, but also can serve as an atypical ATG8-interacting motif (AIM) to selectively recognize mammalian ATG8 family proteins. We determine the high-resolution crystal structures of ATG16L1 FIR in complex with FIP200 Claw and GABARAPL1, respectively, and elucidate the molecular mechanism underlying the interactions of ATG16L1 with FIP200 and ATG8 family proteins. To distinguish the precise contribution of FIP200 from ATG8 family proteins for binding to ATG16L1 FIR in autophagy, we develop a unique ATG16L1 mutant that can exclusively interact with ATG8 family proteins but not FIP200. Finally, using relevant cell-based functional assays, we demonstrate that the interaction of ATG16L1 with FIP200 is indispensable for the effective autophagic flux. In conclusion, our findings provide mechanistic insights into the interactions of ATG16L1 with FIP200 and ATG8 family proteins, and are valuable for further understanding the function of ATG16L1 in autophagy.

Biological sciences/Structural biology/X-ray crystallography

Biological sciences/Cell biology/Autophagy/Macroautophagy

Autophagy

ATG16L1

FIP200

GABARAPL1

ATG8 family proteins

WIPI2

Macroautophagy (hereafter referred to as autophagy) is a well-conserved and lysosome-dependent catabolic process that recycles undesired or harmful cytosolic components for providing essential building blocks to maintain cellular homeostasis in mammals^1–4. Autophagy necessitates de novo synthesis of double-membrane autophagosome through the sophisticated cooperation of a series of autophagy-related (ATG) proteins^3,5,6. During the amino acid starvation-induced canonical autophagy, calcium transients trigger liquid-liquid phase separation of FAK family kinase-interacting protein of 200 kDa (FIP200) and the FIP200-containing Unc-51-like kinase (ULK) complex in the pre-autophagosomal structure (PAS) to initiate autophagosome formation⁷. Concurrently, the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is translocated to the PAS, and is subsequently activated to phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol-3-phosphate (PI3P)⁸. Meanwhile, both the FIP200 subunit of the ULK complex and WD repeat domain phosphoinositide-interacting protein 2 (WIPI2) that senses the PI3P signal from PI3KC3-C1 can regulate the targeting of the E3-like ATG12 ~ ATG5-ATG16L1 complex (the ATG16L1 complex) to the PAS^9–12. Subsequently, the ATG16L1 complex catalyzes the phosphatidylethanolamine (PE) lipidation of ATG8 family proteins to facilitate the elongation of PAS to form autophagosome^13–15. Although the molecular mechanism underpinning the recruitment of the ATG16L1 complex by WIPI2 is well elucidated in previous studies^12,16, how FIP200 specifically interacts with ATG16L1 to recruit the ATG16L1 complex remains elusive. Moreover, why autophagy deploys two distinct approaches to recruit the ATG16L1 complex is still enigmatic.

In addition to non-selective canonical autophagy, accumulating studies have uncovered considerable selective autophagy processes mediated by different types of autophagy receptors^17–23. Currently identified autophagy receptors in mammals, such as p62, NBR1, Optineurin, CCPG1, NDP52 and TAX1BP1, all encompass a cargo-associating domain that can specially recognize certain types of autophagic cargoes as well as a unique ATG8-interacting motif (AIM) that recognizes the ATG8 family proteins known as LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2 in mammals^{19,20,24–26}. Moreover, recent studies revealed that in order to induce in situ autophagosome formation to envelope and sequester the targeting cargoes, many autophagy receptors can directly interact with FIP200 to recruit the ULK complex^27–31. Particularly, some autophagy receptors can directly bind to the Claw domain of FIP200 through their respective FIP200-interacting region (FIR) for recruiting the ULK complex^28,29,31,32. Since the sequence pattern of AIM bears a striking resemblance to that of FIR, the AIM motifs of many autophagy receptors can also function as FIR to interact with FIP200 Claw^28,29. However, due to the high similarity, it is challenging to dissect the individual contribution of FIP200 and ATG8 family proteins for binding to these autophagy receptors. Interestingly, similar to aforementioned autophagy receptors, mammalian ATG16L1 can function as an adaptor to specifically recognize invading pathogens or pathogen-containing vacuoles through its C-terminal WD40 repeats domain to mediate the antibacterial selective autophagy (also named as xenophagy)^33–36. Moreover, a previous elegant study of the network organization of the human autophagy system well demonstrated that ATG16L1 can directly bind to ATG8 family proteins³⁷. However, how ATG16L1 interacts with ATG8 family proteins and the detailed relationship between FIP200 and ATG8 family proteins in binding to ATG16L1 are largely unknown.

In this study, we discover that ATG16L1 contains a typical FIR motif for directly interacting with the Claw domain of FIP200. Importantly, in addition to binding to FIP200 Claw, ATG16L1 FIR can also serve as a noncanonical AIM motif to selectively recognize mammalian ATG8 family proteins. Moreover, our determined crystal structures of the ATG16L1 FIR/FIP200 Claw complex and the ATG16L1 FIR/GABARAPL1 complex not only uncover the detailed molecular mechanism governing the specific interactions of ATG16L1 with FIP200 and ATG8 family members, but also reveal that FIP200 and ATG8 family proteins are mutually exclusive in binding to ATG16L1. On this basis, we devise a specific ATG16L1 mutant that can well interact with ATG8 family proteins but not FIP200. Finally, using this ATG16L1 mutant together with relevant cell-based functional assays, we demonstrate that the interaction of ATG16L1 FIR with FIP200 is indispensable for the effective autophagic flux in canonic autophagy.

Biochemical characterizations of the interaction of ATG16L1 with FIP200 Claw

Previous studies uncovered that the ATG16L1(229–242) region adjacent to the WIPI2-binding site 1 (WBS1) of ATG16L1 is essential for the interaction between ATG16L1 and FIP200 (Fig. 1a)^9,10. Consistently, using size exclusion chromatography (SEC)-based assays, we demonstrated that the highly conserved central region of ATG16L1, the ATG16L1(78–247) fragment (Supplementary Fig. 1), can readily interact with FIP200(1490–1594) (hereafter referred to as FIP200 Claw) (Fig. 1b). Further multi-angle light scattering analysis revealed that the purified ATG16L1(78–247)/FIP200 Claw complex forms a stable 2:2 stoichiometric complex in solution (Fig. 1c). Strikingly, detailed sequence alignment analysis of the FIP200-binding region of ATG16L1 with that of NAP1, SINTBAD, CCPG1 and NDP52, all of which were demonstrated to directly interact with FIP200 Claw^28,32,38,39, unraveled that the ATG16L1(235–247) region conforms to the criteria for a FIP200 Claw-binding FIR motif²⁸, likely representing a putative FIR motif (hereafter referred to as ATG16L1 FIR) (Fig. 1d). Indeed, further quantitative fluorescent polarization (FP)-based assay revealed that ATG16L1 FIR can specifically interact with FIP200 Claw with a Kd value of about ~ 1.33 µM (Fig. 1e). In contrast, the removal of ATG16L1 FIR totally disrupted the association of ATG16L1(78–247) with FIP200 Claw (Fig. 1f). Taken together, all these data clearly demonstrated that ATG16L1 contains a conserved FIR motif to directly interact with the Claw domain of FIP200.

The overall structure of the ATG16L1 FIR/FIP200 Claw complex

Then, we intended to determine the ATG16L1 FIR/FIP200 Claw complex structure to uncover how ATG16L1 FIR binds to the Claw domain of FIP200. After numerous attempts, we finally managed to solve the crystal structure of ATG16L1 FIR in complex with FIP200 Claw at 1.61 Å resolution (Supplementary Table 1). The determined ATG16L1 FIR/FIP200 Claw complex structure is composed of two ATG16L1 FIR molecules and a FIP200 Claw dimer, forming a unique 2:2 stoichiometric hetero-tetramer (Fig. 2a), in line with our aforementioned multi-angle light scattering result (Fig. 1c). In the complex structure, except for a distinct N-terminal α-helix that was only found in one of the two FIP200 Claw domains owing to crystal packing (Supplementary Fig. 2a), the two monomeric FIP200 Claw domains adopt a highly similar core architecture assembled by six β-strands and one α-helix (Supplementary Fig. 2b). In parallel, the two ATG16L1 FIR molecules in the complex structure mainly form two short β-strands that symmetrically augment the β4-strand of two FIP200 Claw domains in an anti-parallel manner (Fig. 2a). Further structural comparison analyses showed that the overall structure of the monomeric FIP200 Claw domain in the ATG16L1 FIR/FIP200 Claw complex is highly akin to that of the apo-form FIP200 Claw domain (PDB ID: 6DCE) (Supplementary Fig. 2c), whereas the binding of ATG16L1 FIR to FIP200 Claw induces an obvious conformational rearrangement of the FIP200 Claw dimer (Fig. 2b). Notably, similar phenomena were also observed in our previous studies^28,32.

The binding interface between ATG16L1 FIR and FIP200 Claw

In the ATG16L1 FIR/FIP200 Claw complex structure, each ATG16L1 FIR molecule packs extensively with a highly electropositive and hydrophobic concave groove that is situated adjacent to the β4/β5 connecting region of the monomeric FIP200 Claw, burying a total interface area of ~ 505 Å² (Fig. 2a). Further careful analyses of the molecular interface in the ATG16L1 FIR/FIP200 Claw complex structure revealed that the hydrophobic side chain of ATG16L1 I240 deeply inserts into a hydrophobic pocket formed by the side chains of C1565, A1567, F1574 and F1582 residues of FIP200 Claw (Fig. 2c and Supplementary Fig. 2d), and concurrently, the hydrophobic side chains of ATG16L1 I243 and V244 residues form hydrophobic contacts with the aromatic side chain of Y1564 and the aliphatic side chain group of K1581 from FIP200 (Fig. 2c). Furthermore, the negatively charged D238, D239 and E241 residues of ATG16L1 FIR form specific hydrogen bonding and charge-charge interactions with the positively charged K1569, R1573 and K1568 residues of FIP200 Claw (Fig. 2c and d). In addition, the backbone groups of E241, I243 and V244 residues of ATG16L1 FIR couple with the backbone groups of Q1566 and Y1564 residues of FIP200 to form five specific backbone hydrogen bonds (Fig. 2c). In keeping with their critical structural roles, all these key binding interface residues of ATG16L1 and FIP200 are highly conserved in different eukaryotic species (Supplementary Figs. 1 and 3). Consistent with our structural results, further FP-based assays showed that point mutations of key interface residues of FIP200 Claw, such as the Y1568A, K1569A, R1573E and F1574Q mutations, all significantly decrease the specific interaction of FIP200 Claw with ATG16L1 FIR (Fig. 2e and Supplementary Fig. 4). Reciprocally, point mutations of key interface residues from ATG16L1 FIR, including the D238R, D239R, I240Q, E241R and I243Q mutations of ATG16L1 FIR, all largely attenuate or essentially disrupt the interaction between FIP200 Claw and ATG16L1 FIR (Supplementary Fig. 5). Therefore, all those mutagenesis-based biochemical assays confirmed the specific interaction between ATG16L1 FIR and FIP200 Claw observed in the solved ATG16L1 FIR/FIP200 Claw complex structure.

Biochemical characterizations of the interactions of ATG16L1 with ATG8 family proteins

Since the reported FIR motifs have been demonstrated to directly engage with mammalian ATG8 family proteins^28,32, we suspected that ATG16L1 FIR may also recognize mammalian ATG8 orthologs. Indeed, our ITC analyses revealed that ATG16L1 FIR can directly interact with six mammalian ATG8 members, and preferentially binds to GABARAPL1 and LC3C with the Kd value of ~ 4.59 µM and ~ 6.27 µM, respectively (Fig. 3a, 3f and Supplementary Fig. 6). Then, we chose GABARAPL1 as a representative to further characterize the binding mechanism of ATG16L1 FIR with ATG8 family proteins. Our SEC results showed that the FIR-containing ATG16L1(78–247) and ATG16L1(235–247) fragments can readily interact with GABARAPL1 (Supplementary Fig. 7a and b). In contrast, the ATG16L1(78–235) fragment, which lacks the FIR motif of ATG16L1, is completely unable to interact with GABARAPL1 (Supplementary Fig. 7c). Concurrently, we also utilized NMR to validate the interaction between GABARAPL1 and ATG16L1. Titrations of ¹⁵N-labelled GABARAPL1 with un-labelled ATG16L1(235–247) or ATG16L1(78–247) proteins showed that many peaks in the ¹H-¹⁵N HSQC spectra of GABARAPL1 undergo significant chemical shift changes or peak-broadenings, confirming that the FIR motif-containing ATG16L1 fragments can directly bind to GABARAPL1 (Supplementary Fig. 8). Taken together, all these data clearly demonstrated that ATG16L1 FIR can function as an unconventional AIM motif to directly recognize mammalian ATG8 orthologs.

The structure of ATG16L1 FIR in complex with GABARAPL1

To further uncover the detailed molecular mechanism underlying the selective recognitions of ATG8 family proteins by ATG16L1 FIR, we determined the high-resolution crystal structure of GABARAPLI in complex with ATG16L1 FIR (Supplementary Table 1). In the determined GABARAPL1/ATG16L1 FIR complex structure, GABARAPL1 adopts a typical ATG8 family protein architecture assembled by a ubiquitin-like structural core and two preceding N-terminal α-helices (Fig. 3b). In the complex structure, the clearly defined ATG16L1 FIR motif contains 10 highly conserved residues spanning from Q236 to D245 of ATG16L1 (Supplementary Fig. 9a), and adopts an extended configuration to occupy the canonical AIM-binding groove of GABARAPL1, which is mainly formed by the β1-strand, β2-strand, α2-helix and α3-helix of GABARAPL1 (Fig. 3c-e). Further structural analyses of the binding interface of the GABARAPL1/ATG16L1 FIR complex revealed that the interaction between GABARAPL1 and ATG16L1 FIR is mainly mediated by both polar and hydrophobic interactions (Fig. 3c). In particular, the negatively charged side chains of ATG16L1 D239, E241 interact with the positively charged side chains of K48, K46 and R67 of GABARAPL1 to form three charge-charge interactions, and the backbone amide and oxygen groups of ATG16L1 E241 together with the backbone amide group of ATG16L1 I243 form three backbone hydrogen bonds with the GABARAPL1 K48 and L50 residues (Fig. 3c and d). In addition, the GABARAPL1/ATG16L1 FIR interaction is further strengthened by four specific hydrogen bonds, two of which are formed between the backbone oxygen groups of D238 and I240 residues of ATG16L1 and the positively charged side chain of GABARAPL1 K46, while the other two are mediated by the backbone oxygen group of ATG16L1 I243 and the positively charged side chain of GABARAPL1 R28 (Fig. 3c). In parallel, the hydrophobic side chains of ATG16L1 I240 and V242 residues pack against a hydrophobic groove of GABARAPL1 formed by the hydrophobic side chains of I21, P30, L50, F104 and the aliphatic portion of the side chain of GABARAPL1 K48 residue (Fig. 3c and e). Moreover, the hydrophobic side chain of ATG16L1 I243 occupies a hydrophobic pocket assembled by the side chains of Y49, V51, P52, L55, F60 and L63 residues of GABARAPL1 (Fig. 3c and e). Consistently, all these key binding interface residues of ATG16L1 FIR and GABARAPL1 are highly conserved during evolution (Supplementary Figs. 1 and 10). Using ITC-based assays, we further verified the specific interactions between interface residues of GABARAPL1 and ATG16L1 FIR observed in the complex structure. Individual point mutations of key residues involved in the binding interface of GABARAPL1/ATG16L1 FIR complex either from GABARAPL1 or ATG16L1 FIR, such as the I21Q, K48E, L50Q and R67E mutations of GABARAPL1 (Fig. 3f and Supplementary Fig. 11), or the D239R, I240Q, E241R, V242E and I243Q mutations of ATG16L1 FIR (Fig. 3f and Supplementary Fig. 12), all dramatically decrease or essentially disrupt the association of GABARAPL1 with ATG16L1 FIR. Notably, previous studies showed that the core unconventional AIM motif of NAP1, NDP52 or TAX1BP1 consists of an acidic Asp followed by four consecutive hydrophobic residues, all of which participate in hydrophobic interactions with the relevant ATG8 family proteins^26,32,40. Interestingly, unlike that of the unconventional AIM motifs of NAP1, NDP52 and TAX1BP1, the third residue of the core unconventional AIM motif of ATG16L1 is an acidic Glu residue (Supplementary Fig. 9b), which is directly involved in the interaction with GABARAPL1 (Fig. 3c). Therefore, ATG16L1 FIR represents a unique type of unconventional AIM motif.

The relationships of FIP200, GABARAPL1 and WIPI2 in binding to ATG16L1

Based on our aforementioned structural analyses, ATG16L1 FIR adopts essentially the same key residues to recognize FIP200 Claw and GABARAPL1, such as the D239, I240, E241 and I243 residues (Figs. 2c, 3c and Supplementary Fig. 1). Thus, FIP200 and ATG8 family proteins should be mutually exclusive in binding to ATG16L1 FIR. As expected, further SEC coupled with SDS-PAGE assays confirmed that FIP200 Claw and GABARAPL1 are competitive in binding to ATG16L1 (Fig. 4a and b). Given that ATG16L1 FIR is C-terminally adjacent to the WIPI2-binding site 1 (WBS1) of ATG16L1 (Fig. 1a), we also tested the relationship between WIPI2 and FIP200 or GABARAPL1 in binding to ATG16L1. Intriguingly, we revealed that FIP200 Claw but not GABARAPL1, can form a stable ternary complex with WIPI2 and the ATG16L1(207–247) fragment that contains both WBS1 and FIR (Supplementary Fig. 13a), suggesting that WIPI2 and FIP200 can simultaneously bind to ATG16L1, consistent with a previous study¹¹. In contrast, WIPI2 and ATG8 family protein, such as GABARAPL1, are competitive in binding to ATG16L1(207–247) (Supplementary Fig. 13b), likely due to the potential steric hindrance.

The rational design of selective ATG16L1 mutant for binding to GABARAPL1 but not FIP200

Based on our previous study²⁸, the consensus FIR motif (Ψ-Θ-Χ1-Χ2-Φ, where Ψ represents a phosphorylated Ser/Thr residue or an acidic Asp, Glu, Θ represents a bulk hydrophobic Ile, Leu, Met or aromatic Phe, Tyr, Trp residue, Φ represents a hydrophobic Leu, Ile or Val residue, and Χ1/Χ2 represents any residue), bears a striking resemblance to the sequence pattern of the core AIM sequence. Actually, the similarity between FIR and AIM severely interferes with our assessment of the individual contribution made by FIP200 and ATG8 family proteins when binding to ATG16L1 FIR. Therefore, it is necessary to develop selective ATG16L1 mutants that can exclusively interact with FIP200 or ATG8 family proteins. Based on our previous biochemical and structural characterizations of relevant FIR and AIM motifs^28,41, we realized that the first residue preceding the consensus core sequence of AIM, which is corresponding to the Ψ residue of FIR, can tolerate basic residues to some extent, such as Arg residue, while FIR cannot. In addition, the Θ residue of AIM prefers an aromatic Phe, Tyr, or Trp residue rather than a hydrophobic Ile, Leu, or Val residue. Eventually, we managed to devise a selective ATG16L1 D239R/I240F (DRIF) double mutant that can solely recognize GABARAPL1 but not FIP200 (Fig. 4c-f). In addition, we also obtained a unique ATG16L1 I240Q/I243Q (IQIQ) double mutant, which binds neither GABARAPL1 nor FIP200 (Fig. 4c, d, g, h). Importantly, in agreement with our biochemical data (Fig. 4c-h), further co-immunoprecipitation assays showed that the DRIF double mutant can selectively bind to GABARAPL1 but not FIP200 in cells (Fig. 5a and b). Concomitantly, the IQIQ double mutation of ATG16L1 completely abolishes the specific interactions of ATG16L1 with FIP200 and GABARAPL1 in cells (Fig. 5a and b). Notably, in order to avoid potential ATG8ylation of ATG16L1 in cells⁴², the C-terminal Gly-Lys of GABARAPL1 was removed in these assays. Unfortunately, albeit with numerous attempts, we failed to obtain an ATG16L1 mutant, which can only bind to FIP200 with a comparable binding ability as the wild-type ATG16L1 but not GABARAPL1.

The essential role of ATG16L1 FIR for the effective autophagic flux in canonical autophagy

To further unravel the functional relevance of ATG16L1 FIR in autophagy, we back-transfected the ATG16L1 knockout HeLa cell line, which was generated in our previous study¹², with relevant plasmids to stably express the wild-type ATG16L1 or relevant ATG16L1 mutants, such as the DRIF mutant of ATG16L1 that only loses the FIP200-binding ability, or the ATG16L1 IQIQ mutant that simultaneously loses the abilities for interacting with FIP200 and ATG8 family proteins. In line with our previous study¹², the autophagic flux in the ATG16L1 knockout cells was effectively rescued by the wild-type ATG16L1. However, both ATG16L1 DRIF and ATG16L1 IQIQ mutants only partially restored amino acid starvation-induced LC3B lipidation, and were essentially unable to recover the autophagic degradation of p62 (Fig. 5c and d), underscoring an indispensable role of ATG16L1 FIR in starvation-induced autophagy. Taken together, these cell-based functional data clearly demonstrated that the interaction between ATG16L1 FIR and FIP200 is essential for the effective autophagic flux in canonical autophagy.

In this work, we uncovered that in addition to interacting with FIP200 Claw, ATG16L1 FIR can also serve as an unconventional AIM to recognize ATG8 family proteins. Furthermore, our biochemical and structural analyses revealed that ATG16L1 FIR can selectively bind to six mammalian ATG8 family proteins, but preferentially bind to GABARAPL1 and LC3C (Fig. 3f and Supplementary Fig. 6). Of note, detailed sequence alignment analysis elucidated that several key interface residues for interacting with ATG16L1 are quite different among six mammalian ATG8 family proteins (Supplementary Fig. 14). For instance, the residue corresponding to the R28 residue of GABARAPL1 that forms two hydrogen bonds with the backbone oxygen group of ATG16L1 I243, is a Lys residue in the LC3 subfamily; the residues corresponding to the bulk hydrophobic L55 and F60 residues in GABARAPL1 are two much smaller Val and Leu residues in LC3A and LC3B (Supplementary Fig. 14). The identification of these non-conserved interface residues among different ATG8 family proteins is likely to rationalize the selective recognition of different mammalian ATG8 orthologs by ATG16L1 FIR. Notably, based on this study together with previous reports^28,32,40, the X1 position of FIR or AIM can accommodate acidic Glu residue as well as hydrophobic residues, including Ile, Val and Cys (Fig. 1d), for engaging with FIP200 or ATG8 family proteins. It is noteworthy that the X1 residues in the FIR and/or AIM of SINTBAD and p62 are Ser/Thr residues that might undergo phosphorylation (Fig. 1d). Accordingly, whether there might be a potential regulatory role of X1 phosphorylation in tuning the interactions of SINTBAD, p62 or other related autophagic factors with FIP200 and ATG8 family proteins remains an open question that is worthwhile to be addressed in the future.

Based on our solid biochemical and structural results (Figs. 2 and 3), ATG16L1 FIR adopts many identical interface residues to interact with FIP200 Claw and ATG8 family proteins. Therefore, the simple deletion or mutagenesis of ATG16L1 used to disrupt the ATG16L1/FIP200 interaction in previous functional studies actually leads to the loss of the interactions of ATG16L1 with both FIP200 and ATG8 family proteins^9–11,36. Consequently, the exact function of FIP200 for binding to ATG16L1 in autophagy remained unclear in previous studies. In this study, by developing and utilizing the ATG16L1 DRIF mutant that solely loses its ability to interact with FIP200 but not ATG8 family proteins, we confidently elucidated that the ATG16L1/FIP200 interaction is essential for the amino acid starvation-induced canonical autophagy. Given that the FIR/AIM motif exhibits universally in many autophagy receptors^28,29, the DRIF mutation strategy utilized for ATG16L1 in this study might also be employed to evaluate the respective roles of FIP200 and ATG8 family proteins in binding to relevant autophagy receptors during selective autophagy.

Previous functional studies well established that mammalian ATG16L1 can function as an adaptor to specifically recognize invading pathogens or pathogen-containing vacuoles through its C-terminal WD40 repeats domain to mediate xenophagy processes^33,34,36,43. Meanwhile, when ectopically targeted to the mitochondria, a fragment containing the ATG16L1 FIP200-binding region is sufficient to initiate de novo autophagosome biogenesis through the recruitment of FIP200-containing ULK complex³⁰. Intriguingly, the deficiency of FIP200 blocks mitophagy rather than ATG16L1-mediated xenophagy, implying the dispensable role of the FIP200/ATG16L1 interaction in ATG16L1-mediated xenophagy³⁴. Based on our discovery that FIP200 and ATG8 family proteins are mutually exclusive in binding to ATG16L1 FIR, we suspected that during ATG16L1-mediated xenophagy, once the ATG16L1 complex is recruited onto the bacteria-containing vacuole, ATG16L1 FIR is likely to be rapidly saturated by surrounding excessive lipidated ATG8 family proteins, thereby depriving of the opportunity of ATG16L1 to interact with FIP200. In contrast, the mitochondria-localized ATG16L1 FIP200-binding fragment lacks E3-like enzyme activity to generate PE-conjugated ATG8 family proteins in the vicinity, thereby enabling its recruitment of the ULK complex via binding to FIP200. Unfortunately, the lack of specific ATG16L1 mutant, which selectively binds to FIP200 but not ATG8 family proteins, precludes further evaluations of our hypothesis.

Notably, p62-ubiquitin condensates recruit the ULK complex upon the interaction between p62 FIR and FIP200 Claw, thereby promoting the autophagy-dependent degradation of ubiquitinated cargoes²⁷. In this study, our biochemical and structural data demonstrated that ATG16L1 FIR is directly involved in the interaction with FIP200 Claw (Figs. 1e, 2 and 4d). Thus, the deletion of FIP200 Claw not only abolishes the association between p62 and FIP200, but also eliminates the interaction of FIP200 with ATG16L1 as well as many other autophagy receptors, such as TAX1BP1 that can indirectly associate with p62 through NBR1⁴⁴. Therefore, the related functional data derived from the deprivation of FIP200 Claw in the previous study should be interpreted with caution²⁷.

In mammals, ATG16L1 is equipped with two distinct WIPI2-binding sites and one FIP200-interacting region to facilitate the normal progression of autophagy^9,10,12. In contrast, ATG16 in yeast encompasses only one conserved site to bind ATG21, the yeast homologue of WIPI2^45,46. In addition, the direct interaction between the ATG1 complex and the ATG16 complex is mediated by the N-terminal segment of yeast ATG12 rather than mammalian ATG16L1 FIR⁴⁷. Thus, although the detailed binding mechanisms are different, the associations of the ATG16/ATG16L1 complex with the ATG1/ULK complex and ATG21/WIPI2 in canonical autophagy are well conserved from yeast to mammals. Based on our functional data in this study, the ATG16L1 FIR/FIP200 interaction is essential for starvation-induced canonical autophagy (Fig. 5c and d). Therefore, the expeditious autophagosome biogenesis in canonical autophagy is unlikely to be adequately explained by merely two distinct linear hierarchical pathways started from the ULK complex. Accordingly, we proposed a positive feedback loop model to illustrate the function of ATG16L1 in canonical autophagy. In this model, the FIP200-containing ULK complex at PAS initiates the translocation of the PI3KC3-C1 complex (Fig. 5e). Subsequently, the activated PI3KC3-C1 complex results in the redistribution of WIPI2 through the generation of substantial PI3P molecules (Fig. 5e). Whereafter, WIPI2 recruits and activates the ATG16L1 complex, which inversely promotes the recruitment of additional ULK complexes to PAS through a binding mechanism between ATG16L1 FIR and FIP200 Claw as uncovered in this study, thereby facilitating the rapid expansion and closure of phagophore membrane to form autophagosome (Fig. 5e).

Materials.

HEK293T and HeLa cell lines were kindly provided by Prof. Junying Yuan from Interdisciplinary Research Center on Biology and Chemistry, CAS, Shanghai, China. The full length human WIPI2b, ATG8s, ATG16L1 and FIP200 genes were obtained from Prof. Jiahuai Han from School of Life Sciences, Xiamen University, Xiamen, China. The synthetic peptide "EQDDDIEVIVDET" (ATG16L1 FIR) was purchased from the China Peptides company, and the purity of the commercially synthesized peptides was > 98%.

Protein expression and purification.

The DNA fragments encoding human FIP200 (residues 1490–1594) and WIPI2b (residues 13–362 without 265–297) were cloned into pET-SUMO-3C vector or pET-32M-3C vector (modified versions of pET-32a vector containing an N-terminal SUMO or Trx tag). The DNA fragments encoding ATG16L1 (residues 207–247, 78–235, 235–247 and 78–247) were all cloned into pACYC-Trx1-3C vector (a modified version of pACYC vector containing an N-terminal Trx tag). Meanwhile, the DNA fragments encoding six human ATG8s were all cloned into pET-32M-3C vector or pET-GST-3C vector (modified versions of pET-32a vector containing an N-terminal GST tag). Of note, 6xHis was placed on either the N-terminal or the C-terminal of target proteins. For Co-immunoprecipitation assays, the DNA fragments encoding human GABARAPL1(1-115) and full-length FIP200 were separately cloned into pEGFP-C1 vector and pmEGFP-C1 vector (a modified version of pEGFP-C1 with an A206K mutation that monomerizes EGFP) as well as full-length ATG16L1 into pFlag-CMV-2 vector. All point mutations of WIPI2b, FIP200, GABARAPL1 and ATG16L1 used in this study were generated through standard PCR-based mutagenesis method and further confirmed by DNA sequencing.

Recombinant proteins were all expressed in BL21 (DE3) E. coli cells induced by 200 µM IPTG overnight at 16°C. The bacterial cell pellets were re-suspended in the binding buffer (50 mM Tris, 500 mM NaCl, and 5 mM imidazole at pH 7.9), and then lysed by the ultrahigh pressure homogenizer FB-110XNANO homogenizer machine (Shanghai Litu Machinery Equipment Engineering Co., Ltd.). Then, the lysate was spun down by centrifuge at 17000 rpm (35000 g) for 35 minutes to remove the pellets fractions. All proteins were purified by Ni²⁺-NTA agarose (GE Healthcare) affinity chromatography and further purified by size-exclusion chromatography (Superdex 75 or 200 26/60 column; GE Healthcare) equilibrated with the column buffer containing 20 mM Tris, 100 mM NaCl, 1 mM DTT and 1 mM EDTA at pH 7.5. To obtain the FIP200 Claw/ATG16L1 FIR complex used for crystallization, the FIP200/ATG16L1 complex was obtained through the co-expression of Trx-FIP200(1490–1594) and Trx-ATG16L1(235–247). The N-terminal Trx tags of relevant FIP200 and ATG16L1 proteins were cleaved by 3C protease and removed by HisTrap excel column (GE Healthcare). Finally, the FIP200 Claw/ATG16L1 FIR complex and the GABARAPL1/ATG16L1 FIR complex were further purified through Superdex 75 column equilibrated with the aforementioned column buffer. Special for six human ATG8 family proteins, their Trx tags were removed by MonoQ 10/10 ion-exchange column (GE Healthcare). Meanwhile, uniformly ¹⁵N-labelled GABARAPL1 proteins were prepared by growing bacteria in M9 minimal medium using ¹⁵NH₄Cl (Cambridge Isotope Laboratories Inc.) as the sole nitrogen source.

ITC assay.

ITC measurements were all carried out on a MicroCal PEAQ-ITC (Malvern) calorimeter at 25°C. All protein samples were prepared in the same buffer containing 20 mM Tris, 100 mM NaCl, 1 mM DTT at pH 7.5. For each ITC experiment in this study, the concentrated (~ 50 µM) proteins were loaded into the cell, and the other titrated proteins (~ 500 µM) were loaded into the syringe. The titration processes were performed by injecting proteins from syringe into the cell at time intervals of 2 minutes to ensure that the titration peak returned to the baseline. The titration data were analyzed using the Malvern MicroCal PEAQ-ITC analysis program and fitted using the one-site binding model.

Size exclusion chromatography.

Size exclusion chromatography was carried out on an AKTA FPLC system (GE Healthcare). Purified proteins were loaded on to a Superdex 200 or 75 increase 10/300 GL column (GE Healthcare) equilibrated with the same column buffer. The fitting results were further output to the Origin 9 software and aligned with each other.

NMR spectroscopy.

The ¹⁵N-labelled protein samples for NMR titration experiments were concentrated to ~ 0.1 mM. All the protein samples for NMR studies were prepared in the 50 mM potassium phosphate buffer containing 100 mM NaCl and 1 mM DTT at pH 6.5, and NMR spectra were acquired at 25°C on an Agilent 800 MHz spectrometer equipped with an actively z gradient shielded triple resonance cryogenic probe at the Shanghai Institute of Organic Chemistry.

Multi-angle light scattering.

For multi-angle light-scattering measurement, the FIP200(1490–1594)/ATG16L1(78–247) complex samples were injected into an AKTA FPLC system (GE Healthcare) with a Superdex 200 increase 10/300 GL column (GE Healthcare) with the same column buffer mentioned before. The chromatography system was coupled to a static light scattering detector (miniDawn, Wyatt Technology) and a differential refractive index detector (Optilab, Wyatt Technology). Data were collected every 0.5 s with a flow rate of 0.5 mL/min. Data were analyzed using the ASTRA 6 software (Wyatt Technology) and drawn using the Origin 9 software.

Fluorescence polarization assay.

Fluorescence anisotropy binding assays were performed on the SpectraMax i3x Multi-Mode Detection Platform from Molecular Devices, using a 485 nm excitation filter and a 535 nm emission filter. Peptides were labeled with fluorescein isothiocyanate isomer I (Sigma-Aldrich) at their N-terminal NH₂. In this assay, the FITC-labeled peptide (~ 0.3 µM) was titrated with increasing amount of testing proteins in the column buffer at 25 °C. The Kd values were obtained by fitting the titration curves with the classical one-site binding model using GraphPad Prism 9 software.

Protein crystallization and structural elucidation.

Crystals of the FIP200(1490–1594)/ATG16L1(235–247) complex and the GABARAPL1/ATG16L1(235–247) complex were all obtained using the sitting-drop vapor-diffusion method at 16°C. The crystal-growing condition of the FIP200(1490–1594)/ATG16L1(235–247) complex (38 mg/mL) contains 10% v/v 2-Propanol, 0.1 M BICINE (pH 8.5), and 30% w/v Polyethylene glycol 1500. As for the GABARAPL1/ATG16L1(235–247) complex, the purified GABARAPL1 protein (27 mg/mL) was saturated with ATG16L1 FIR peptide with a molar ratio up to 1:10. Crystals were observed in the condition containing 0.1 M Sodium cacodylate (pH 6.5), 40% v/v MPD and 5% w/v PEG 8000. Before diffraction experiments, relevant amount of glycerol was added as the cryo-protectant. A 1.76 Å resolution X-ray data set for the FIP200(1490–1594)/ATG16L1(235–247) complex and a 1.54 Å resolution X-ray data set for the GABARAPL1/ATG16L1(235–247) complex were collected at the beamline BL19U1, BL02U1 and BL10U2 of the Shanghai Synchrotron Radiation Facility⁴⁸. The diffraction data were processed upon autoPROC⁴⁹. The phase problems of the FIP200/ATG16L1 complex and the GABARAPL1/ATG16L1 complex were all solved by molecular replacement method by using the FIP200 Claw structure (PDB ID: 6DCE) and the GABARAPL1 structure (PDB ID: 5LXI) respectively as the search model with PHASER⁵⁰. The initial structural models were rebuilt manually using COOT⁵¹, and then refined through PHENIX⁵². Further manual model building and adjustments were completed via COOT ⁵¹. The qualities of the final models were validated by MolProbity⁵³. The final refinement statistics of solved structures in this study were listed in Supplementary Table 1. All the structural diagrams were prepared using the program PyMOL (http://www.pymol.org/).

Co-immunoprecipitation assay.

Flag-tagged ATG16L1 plasmids (wild-type or mutants) were co-transfected into HEK293T cells using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) or Lipofectamine 6000 transfection reagent (Beyotime) with mEGFP-tagged FIP200 or EGFP-tagged GABARAPL1(1-115) plasmids. Cells were collected 24 hours after transfection and lysed in ice-cold cell lysis buffer (50 mM Tris, 150 mM NaCl, 0.5% NP-40, 1 mM PMSF, 1% protease inhibitor cocktail at pH 7.5) for 20 to 40 minutes at 4°C. Lysates were centrifuged at 14500 g for 15 minutes at 4°C to separate soluble fractions and cell debris. Supernatants were applied to anti-GFP mAb-Agarose (Medical & Biological Laboratories) and incubated for 40 to 60 minutes at 4°C. The beads and non-bound proteins were separated by centrifugation at 800 g for 3 minutes at 4°C. After washing several times with the cold wash buffer (50 mM Tris, 150 mM NaCl and 0.1% or 0.5% NP-40 at pH 7.5), the beads were re-suspended with the 1X SDS-PAGE sample buffer and boiled at 65°C for 10 minutes. The prepared samples were analyzed by SDS-PAGE. The EGFP-tagged GABARAPL1(1-115), mEGFP-tagged FIP200 and Flag-tagged ATG16L1 were detected by western blot using the anti-GFP (Proteintech, 50430-2-AP, 1:1000 dilution), anti-GFP (Proteintech, 66002-1-Ig, 1:2000 dilution), anti-Flag (Proteintech, 20543-1-AP, 1:1000 dilution) and anti-Flag (Proteintech, 66008-4-Ig, 1:2000 dilution) primary antibodies.

Generation of relevant ATG16L1 stable cell lines.

The AcGFP1-tagged mutant ATG16L1 was cloned into the pMSCV-blasticidin vector and was co-transfected into HEK293T cells with VSV-G and gag/pol using Lipofectamine 6000 transfection reagent (Beyotime). Notably, the sgRNA-targeting region of ATG16L1 at pMSCV-blasticidin vector was synonymously mutated to avoid being targeted again by Cas9 enzyme. ATG16L1-knockout cells were incubated with polybrene (Sigma-Aldrich) and concentrated virus-containing medium filtered through a 0.45-µm-pore syringe filter. Transfected cells were treated with blasticidin (5 µg/mL; InvivoGen) to generate stable polyclonal cell lines.

Autophagy induction

The ATG16L1-knockout HeLa cells, which were generated in our previous study¹², were rescued by lentiviral transduction with AcGFP1-tagged WT ATG16L1, ATG16L1 D239R/I240F mutant (DRIF), or ATG16L1 I240Q/I243Q mutant (IQIQ). Rescued HeLa cells were separately seeded on a six-well plate. The following day, cells were incubated for 4 hours with DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific), amino acid starvation medium (BOSTER), and amino acid starvation medium with bafilomycin A1 (Selleck) at 400 nM. After starvation treatment, cells were resuspended with the 1X SDS-PAGE sample buffer and boiled for 7 minutes at 100°C. The samples were detected by Western blot using specific ATG16L1 antibody (1:1000; Abcam, catalog no. ab187671), LC3B antibody (1:1000; Abcam, catalog no. ab192890), β-actin antibody (1:5000; Proteintech, catalog no. 66009-1-lg), and p62 antibody (1:1000; Cell Signaling Technology, catalog no. #39749). The data are presented as means ± SEM from three independent experiments. Statistical analyses were performed in GraphPad Prism 9 by two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test, and P value style is P = 0.1234 (not significant (ns)), *P = 0.0332, **P = 0.0021, ***P = 0.0002, and ****P < 0.0001.

Data availability. The coordinates and structure factors of the FIP200 Claw/ATG16L1 FIR complex and the GABARAPL1/ATG16L1 FIR complex solved in this study have been deposited in the Protein Data Bank under the accession code 9J54 and 9JF2, respectively. All additional experimental data are available from the corresponding author on request.

Acknowledgements

We thank SSRF BL19U1, BL02U1 and BL10U2 for X-ray beam time, Prof. Jiahuai Han for the full length WIPI2b, ATG8s, FIP200 and ATG16L1 cDNA. This work was supported by grants from the National Natural Science Foundation of China (32071219, 92253301, 21822705), the CAS Youth Interdisciplinary Team (JCTD-2022-10), the National Basic Research Program of China (2022YFC2303102) (to L.P.), and the Postdoctoral Fellowship Program of China Postdoctoral Science Foundation (GZC20232860) (to X.G.).

Author contributions

X.G. and L.P. designed research; X.G. Y.Z., Y.W., Y.T., H.L., X.Z., Y.Z., H.G., and Z.G. performed research; X.G., and L.P. analyzed data; X.G. and L.P. wrote the paper.

Competing interests

The authors declare no competing interests.

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Molecular bases of the interactions of ATG16L1 with FIP200 and ATG8 family proteins

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Abstract

Figures

Introduction

Results

Biochemical characterizations of the interaction of ATG16L1 with FIP200 Claw

The overall structure of the ATG16L1 FIR/FIP200 Claw complex

The binding interface between ATG16L1 FIR and FIP200 Claw

Biochemical characterizations of the interactions of ATG16L1 with ATG8 family proteins

The structure of ATG16L1 FIR in complex with GABARAPL1

The relationships of FIP200, GABARAPL1 and WIPI2 in binding to ATG16L1

The rational design of selective ATG16L1 mutant for binding to GABARAPL1 but not FIP200

The essential role of ATG16L1 FIR for the effective autophagic flux in canonical autophagy

Discussion

Materials and Methods

Autophagy induction

Declarations

References

Additional Declarations

Supplementary Files

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