Decoding three distinct states of the Syntaxin17 SNAREmotif in mediating autophagosome–lysosome fusionYing Lia, Xiaofang Chenga, Miao Lia, Yingli Wanga

, Tao Fua, Zixuan Zhoua, Yaru Wanga, Xinyu Gonga,

Xiaolong Xua, Jianping Liua, and Lifeng Pana,b,1

aState Key Laboratory of Bioorganic and Natural Products Chemistry, Center for Excellence in Molecular Synthesis, University of Chinese Academy ofSciences, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 200032 Shanghai, China; and bSchool of Chemistry and Material Sciences,Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 310024 Hangzhou, China

Edited by Yihong Ye, NIH, Bethesda, MD, and accepted by Editorial Board Member Axel T. Brunger July 15, 2020 (received for review April 13, 2020)

Syntaxin17, a key autophagosomal N-ethylmaleimide–sensitivefactor attachment protein receptor (SNARE) protein, can associatewith ATG8 family proteins SNAP29 and VAMP8 to facilitate themembrane fusion process between the double-membraned auto-phagosome and single-membraned lysosome in mammalian mac-roautophagy. However, the inherent properties of Syntaxin17 andthe mechanistic basis underlying the interactions of Syntaxin17with its binding proteins remain largely unknown. Here, usingbiochemical, NMR, and structural approaches, we systemicallycharacterized Syntaxin17 as well as its interactions with ATG8family proteins, SNAP29 and VAMP8. We discovered that Syn-taxin17 alone adopts an autoinhibited conformation mediatedby a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE regionof Syntaxin17 contains one LC3-interacting region (LIR) motif,which preferentially binds to GABARAP subfamily members. Im-portantly, the GABARAP binding of Syntaxin17 can release itsautoinhibited state. The determined crystal structure of the Syn-taxin17 LIR–GABARAP complex not only provides mechanistic in-sights into the interaction between Syntaxin17 and GABARAP butalso reveals an unconventional LIR motif with a C-terminally ex-tended 310 helix for selectively binding to ATG8 family proteins.Finally, we also elucidated structural arrangements of the autopha-gic Syntaxin17–SNAP29–VAMP8 SNARE core complex, and uncov-ered its conserved biochemical and structural characteristicscommon to all other SNAREs. In all, our findings reveal three distinctstates of Syntaxin17, and provide mechanistic insights into theSyntaxin17-mediated autophagosome–lysosome fusion process.

autophagy | SNARE | Syntaxin17 | GABARAP |autophagosome–lysosome fusion

Macroautophagy (hereafter referred to as autophagy) relieson the double-membraned vesicle called the autophago-

some to fuse with the lysosome, forming the autolysosome fordegradation of enclosed cytoplasmic materials in eukaryotes(1–5). Through autophagy, eukaryotic cells can recycle macro-molecular constituents, such as bulk protein aggregates, glycogen,dysfunctional organelles, and invading pathogens, to maintaincellular homeostasis and/or adapt to multiple cellular stresses (1,2). Thereby, autophagy plays critical roles in numerous physio-logical processes, such as energy metabolism, immune response,embryogenesis, and aging (6–8). Dysfunctions of autophagy areassociated with many human diseases, including cancer, immunedisorders, and neurodegenerative diseases (8–11). During theautophagy pathway, the formation of the autolysosome representsone of the essential steps for ultimate autophagic degradation, anddepends on the tight coordination of autophagic vesicle fusions (1,4–6, 12). So far, many proteins have been identified as being in-volved in these processes in mammals, including autophagicN-ethylmaleimide–sensitive (NSF) factor attachment protein re-ceptor (SNARE) proteins (13, 14), relevant tethering factors suchas the HOPS complex, ATG14, and EPG5 (15–18), ATG8 familyproteins (19–21), and related regulatory proteins including Rab7,

RILP, TECPR1, PLEKHM1, BRUCE, and Pacer (22–27).However, the detailed molecular mechanisms underlying the co-operation of these proteins to promote the formation of theautolysosome are still not well-understood.Cellular membrane fusion processes are known to be medi-

ated by SNARE proteins, which can assemble into a membrane-bridging four-helix bundle (composed of Qa-, Qb-, Qc-, andR-SNAREs) to provide the mechanical thrust for effectivelydriving membrane fusion (28). The fusion event between thedouble-membraned autophagosome and the single-membranedlysosome during autophagy in mammals is reported to be me-diated by two autophagy-specific SNARE complexes, the Syn-taxin17 (hereafter called STX17)–SNAP29–VAMP8 SNAREcomplex and the recently discovered YKT6–SNAP29–Syntaxin7SNARE complex (13, 14). As the key player for promotingautophagosome–lysosome fusion, the STX17-containing SNAREcomplex is composed of the autophagosomal SNARE STX17(Qa-SNARE), the cytosolic SNARE SNAP29 (Qbc-SNAREs), andthe lysosomal SNAREVAMP8 (R-SNARE) (Fig. 1A). Structurally,SNAP29 mainly contains Qb- and Qc-SNARE motifs, whileVAMP8 is composed of an R-SNARE motif followed by a trans-membrane domain (Fig. 1A). As a Qa-type SNARE protein, STX17contains an N-terminal Habc domain, a Qa-SNARE motif followedby two unique tandem transmembrane domains (Fig. 1A). The two

Significance

Macroautophagy is essential for the maintenance of cellularhomeostasis and physiology in mammals, and relies on vesiclefusion between the autophagosome and the lysosome, formingthe autolysosome to degrade unwanted cytosolic contents forrecycling. The membrane fusion between the autophagosomeand lysosome requires ATG8 family proteins and autophagy-related SNARE proteins including Syntaxin17, VAMP8, andSNAP29, but with poorly understood mechanisms. In this study,through systemic biochemical and structural characterizations,we reveal three different states of the key autophagosomalSNARE protein Syntaxin17 and provide mechanistic insights intothe autoinhibited state of Syntaxin17 as well as its interactionswith ATG8 family proteins, SNAP29 and VAMP8. Our findingsare valuable for further understanding the functions of Syn-taxin17 in the autophagosome–lysosome fusion process.

Author contributions: Y.L. and L.P. designed research; Y.L., X.C., and M.L. performedresearch; Yingli Wang contributed new reagents/analytic tools; Y.L., X.C., M.L., YingliWang, T.F., Z.Z., Yaru Wang, X.G., X.X., J.L., and L.P. analyzed data; and Y.L., X.C., andL.P. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission. Y.Y. is a guest editor invited by theEditorial Board.

Published under the PNAS license.1To whom correspondence may be addressed. Email: panlf@sioc.ac.cn.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2006997117/-/DCSupplemental.

First published August 19, 2020.

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Fig. 1. NMR-based characterizations of the interaction between the STX17 Qa-SNARE motif and its N-terminal Habc domain. (A) A schematic diagramshowing the domain organizations of STX17, SNAP29, VAMP8, and mammalian ATG8 family protein. In this drawing, the boundaries of the relevant domains,motifs, as well as protein fragments of STX17, SNAP29, and VAMP8 used in this study are further labeled, and the interaction between the LIR motif of STX17and the ATG8 family protein is also highlighted and indicated by a two-way arrow. (B) Superposition plot of the assigned 1H-15N HSQC spectra of 15N-labeledSTX17(142–228) titrated with increasing molar ratios of unlabeled STX17(1–123). (C) Plot of backbone amide chemical shift differences and peak broadeningas a function of the residue number of STX17(142–228) between the wild type and the protein titrated with STX17(1–123) at a molar ratio of 1:1. In thisrepresentation, the residues with disappeared NMR peaks due to peak broadening are shown in black and the combined 1H and 15N chemical shift changesare defined as

Δppm = ΔδHN( )2 +   ΔδN × αN( )2[ ]1=2, [1]

where ΔδHN and ΔδN represent differences of the amide proton and nitrogen chemical shifts of each residue of STX17(142–228). The scaling factor (αN) usedto normalize the 1H and 15N chemical shifts is 0.17. (D) Superposition plot of the 1H-15N HSQC spectra of 15N-labeled STX17(1–123) titrated with increasingmolar ratios of unlabeled STX17(142–228). For clarity, the Inset shows an enlarged view of a selected region of the overlaid 1H-15N HSQC spectra. ppm, partsper million.

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transmembrane domains of STX17 are demonstrated to form ahairpin structure and are required for the localization of STX17 onthe autophagosome (13). The Qa-SNARE motif of STX17 cancoassemble with SNAP29 Qb-SNARE, Qc-SNARE motifs, and theR-SNARE motif of VAMP8, forming the SNARE core complex(13), and the structure of this core SNARE complex was deter-mined in a previous study from Zhong’s group (16). However, thebiochemical properties of this autophagic SNARE complex and thedetailed molecular basis underpinning the regulation of this auto-phagic SNARE complex formation are still largely unknown. In-triguingly, in addition to its canonical role in assembling theSNARE core complex, the Qa-SNARE motif of STX17 is alsoimplicated in interactions with many other autophagy-related pro-teins, such as ATG14 (16), TBK1 (29), and ATG8 family proteins(30). In particular, a recent study showed that STX17 can directlyinteract with ATG8 family proteins and IRGM, an autophagy-related small GTPase, through its Qa-SNARE motif region andtwo transmembrane domains, respectively (30). Importantly, theseinteractions of STX17 with ATG8 family proteins and IRGM areessential for the efficient recruitment of STX17 to the autophago-some (30). However, due to the lack of detailed structural charac-terizations, the molecular mechanisms governing the interactions ofSTX17 with these proteins remain elusive.ATG8 family proteins are small ubiquitin-like proteins, and include

six orthologs in mammals known as MAP1LC3A (LC3A),MAP1LC3B (LC3B), MAP1LC3C (LC3C), GABARAP, GABAR-APL1, and GABARAPL2 (31–33). They can be further classifiedinto two subfamilies, the LC3 subfamily and the GABARAP sub-family (31, 32). ATG8s are decorated on the membrane of thephagophore by conjugating with a phosphatidylethanolamine (PE)lipid catalyzed by the E3-like ATG5–ATG12–ATG16L1 complexduring the action of autophagy (4, 5, 32–34). The PE-conjugatedATG8s are present both on the inner and outer membranes of theemerging closed autophagosome before fusion with the lysosome (4,5). They are demonstrated to play crucial roles in autophagosomebiogenesis, autophagic cargo engulfment, autophagic vesicle trans-port, and fusion of the autophagosome with the lysosome or endo-some by associating with relevant proteins that contain a short motifcalled the LC3-interacting region (LIR) in mammals (19, 31–33,35–39). The canonical LIR motif contains a consensus core sequenceΘXXΓ (where Θ represents an aromatic Trp, Tyr, or Phe residue; Γrepresents a bulky hydrophobic Leu, Ile, or Val residue; and Xrepresents any amino acid residue) (31, 38, 40). Additional negativelycharged serine/threonine phosphorylation sites and/or acidic residuespreceding the LIR core sequence are also routinely found in typicalLIR motifs, which are proven to regulate the interactions of LIR-containing proteins with ATG8 family members (31, 38, 40, 41). In-triguingly, our previous study together with other groups’ reportsrevealed that some LIR motifs, such as that of FYCO1, ankyrin-G,and ankyrin-B, also include C-terminal extensions that can participatein the interaction with ATG8 family orthologs following the coreΘXXΓ sequence (42–44). In particular, these C-terminal extensionsnot only can facilitate strong binding to ATG8s but also endow LIR-containing proteins with binding selectivity to different ATG8orthologs (42–44). Notably, previous functional studies well-demonstrated that the GABARAP subfamily is preferentially in-volved in the autophagosome–lysosome fusion process and the LC3sare unable to replace GABARAPs for the fusion between theautophagosome and lysosome (19, 45), suggesting that some proteinsinvolved in the autophagosome–lysosome fusion process may alsocontain C-terminal extensions for selectively binding to theGABARAP subfamily. Interestingly, STX17 was recently reported tocontain two putative LIR motifs within its Qa-SNARE motif region,and can bind to ATG8 family proteins, especially LC3B andGABARAP (30). However, how the two putative LIR motifs ofSTX17 interact with ATG8 family proteins is still elusive.In this study, we biochemically and structurally character-

ized the key autophagosomal SNARE STX17 as well as its

interactions with the ATG8 family proteins, SNAP29 andVAMP8, and uncovered three different states of STX17. Spe-cifically, we discovered that the isolated STX17 adopts anautoinhibited “closed” conformation, in which the N-terminalhalf of the STX17 Qa-SNARE motif occupies the Habc do-main of STX17. In addition, we revealed that STX17 only con-tains one LIR motif, which preferentially binds to GABARAPsubfamily members. We determined the high-resolution struc-ture of the STX17 LIR–GABARAP complex, and uncovered themolecular mechanism underpinning the interaction betweenSTX17 and GABARAP. Notably, the determined STX17 LIR–

GABARAP complex structure also highlights the importance ofthe C-terminal extension following the LIR core motif for someLIR-containing proteins to selectively interact with ATG8 familyproteins. Finally, we also investigated the biochemical andstructural features of the autophagic STX17–SNAP29–VAMP8SNARE core complex, and elucidated characteristic biochemicalproperties and structural arrangements of this autophagic SNAREcomplex.

ResultsThe STX17 SNARE Motif Can Interact with the N-Terminal HabcDomain of STX17 and Alter Its Conformation. To gain molecularinsights into the function of STX17 in the autophagosome–lysosome fusion process, we first conducted a detailed sequencealignment analysis of the cytoplasmic region of STX17 and foundthat the N-terminal Habc region (residues 1 to 123) and the Qa-SNARE motif (residues 167 to 224) of STX17 are highly con-served during evolution (SI Appendix, Fig. S1A), in line with theirknown functions of interacting with other proteins (13, 17).Then, we purified a uniformly 15N-labeled STX17(142–228)fragment that includes the entire Qa-SNARE motif (residues167 to 224), and acquired its 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum (SI Appendix, Fig. S2A).The small dispersion of the NMR peaks in the 1H dimension ofthe 1H-15N HSQC spectrum together with the determined sec-ondary structure of this STX17 Qa-SNARE region based on the13Cα and

13Cβ chemical shift values of each residue after backbonechemical shift assignments indicated that the isolated STX17 Qa-SNARE motif is basically unstructured (SI Appendix, Fig. S2).Interestingly, titration of the 15N-labeled STX17(142–228) withthe unlabeled STX17(1–123) protein showed that a selected set ofpeaks in the 1H-15N HSQC spectrum of STX17(142–228) under-went significant dose-dependent peak broadening or chemicalshift changes (Fig. 1B), indicating that the STX17 Qa-SNAREregion can specifically interact with the N-terminal Habc domainof STX17. Further plotting of the peak broadening and amidebackbone chemical shift changes as a function of residue numberrevealed that the significant perturbations are mainly rich in theN-terminal part of the STX17 Qa-SNARE motif (residues 174 to194) (Fig. 1C), suggesting that this region is the major binding sitefor interacting with the STX17 Habc domain.In contrast to that of STX17(142–228), the 1H-15N HSQC

spectrum of STX17(1–123) is well-dispersed, indicating that thisregion constitutes an independently well-folded domain (Fig. 1D).Further titration of 15N-labeled STX17(1–123) with unlabeledSTX17(142–228) showed that the majority of peaks in the 1H-15NHSQC spectrum underwent significant dose-dependent peakbroadening (Fig. 1D), confirming the existence of a direct inter-action between the Habc domain and Qa-SNARE motif ofSTX17. Strikingly, in the presence of STX17(142–228), a set ofpeaks appeared in the 1H-15N HSQC spectrum of STX17(1–123)(Fig. 1D). Based on this observed NMR phenomenon and a seriesof NMR titration experiments using different truncation mutantsof the STX17 Qa-SNARE motif, we further confirmed that theN-terminal region of the STX17 Qa-SNARE motif is responsiblefor the interaction with STX17(1–123) (SI Appendix, Fig. S3).Unfortunately, due to the serious concentration-dependent peak

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broadening that was likely induced by nonspecific self-associations(SI Appendix, Fig. S4A), we were unable to achieve the backboneassignments for STX17(1–123). However, in the presence of asaturated amount of STX17(142–228) protein, we were able tofinish the backbone assignments for the remaining NMR peaks (SIAppendix, Fig. S4B). Interestingly, we found that the newlyappeared NMR peaks arose from residues 94 to 121, which arelocated in the predicted extreme C-terminal α-helix of the Habcdomain (SI Appendix, Fig. S1A), but were demonstrated to beunstructured in the presence of STX17(142–228) based on ourNMR analysis (SI Appendix, Fig. S4C). Taken together, all thesedata clearly demonstrated that the Qa-SNARE motif of STX17can directly bind to the STX17 Habc domain and alter its con-formation by partially unfolding its extreme C-terminal α-helix.Thus, the cytoplasmic region of STX17 may adopt an autoinhibitedclosed conformation imposed by an intramolecular interaction be-tween its Habc domain and the Qa-SNARE motif. Notably, theSTX17 Qa-SNAREmotif only showed a negligible weak interactionwith the full-length SNAP29 or the VAMP8 R-SNARE motif(residues 8 to 66) proteins based on our NMR titration results (SIAppendix, Fig. S5). Importantly, further NMR analyses revealedthat neither the full-length SNAP29 nor the VAMP8 R-SNAREmotif alone was able to release the autoinhibited state of STX17 (SIAppendix, Fig. S6). Consistent with these NMR-based analyses (SIAppendix, Figs. S5 and S6), further analytical gel filtrationchromatography-based assays revealed that full-length SNAP29cannot obviously interact with VAMP8(1–75) that includes theentire cytoplasmic region of VAMP8 nor with STX17(1–228) (SIAppendix, Fig. S7 A and B), and there is an extremely weak inter-action between STX17(1–228) and VAMP8(1–75) (SI Appendix,Fig. S7C). As expected, when mixing these three proteins together,we can readily detect a ternary SNARE complex containingSNAP29 full-length, VAMP8(1–75), and STX17(1–228) (SI Ap-pendix, Fig. S7 D and E). Therefore, unlike the neuronal SNAREcomplex, the formation of a binary STX17–SNAP29 (Qa/Qbc)t-SNARE complex during the autophagic SNARE complex as-sembly process is unfeasible in vitro.

The STX17 SNARE Region Contains One LIR Motif That Can SelectivelyBind to Mammalian ATG8 Orthologs. Since mammalian ATG8family proteins, especially LC3B and GABARAP, were reportedto interact with the STX17 Qa-SNARE region (30), we wonderedwhether these mammalian ATG8 orthologs might regulate theclosed conformation of STX17. To test this hypothesis, we purifiedtwo STX17 fragments, STX17(142–228) and STX17(1–228), thatinclude the entire cytoplasmic region of STX17 and should adoptan autoinhibited state, and investigated their interactions with thesix mammalian ATG8 homologs. Using analytical gel filtrationchromatography-based analyses, we found both STX17(142–228)and STX17(1–228) can directly interact with all of the six mam-malian ATG8 orthologs (Fig. 2 A and B and SI Appendix, Figs. S8and S9). Further quantitative analyses of the interactions of thesetwo STX17 fragments with different ATG8 homologs using iso-thermal titration calorimetry (ITC) measurements revealed thatSTX17 can selectively bind to six mammalian ATG8 orthologswith distinct binding affinities (Fig. 2 C and D and SI Appendix,Figs. S10–S12). In particular, STX17 preferentially binds toGABARAP subfamily members (GABARAP, GABARAPL1,and GABARAPL2) rather than LC3 subfamily members (LC3A,LC3B, and LC3C) (Fig. 2 C and D and SI Appendix, Figs. S10 andS11). Notably, the STX17(1–228) fragment displays a relativelyweaker binding affinity toward ATG8 family members than that ofSTX17(142–228) based on our ITC analyses (Fig. 2 C and D andSI Appendix, Figs. S10 and S11), suggesting that theSTX17 N-terminal Habc region somehow interferes with the in-teractions between the STX17 SNARE region and ATG8 ortho-logs, consistent with our aforementioned observation that theN-terminal Habc domain of STX17 can directly interact with

the STX17 SNARE motif (Fig. 1 B–D). Importantly, additionalNMR characterizations uncovered that in contrast to SNAP29 andVAMP8 (SI Appendix, Fig. S6), GABARAP can compete againstthe N-terminal Habc domain for binding to the SNARE motif ofSTX17, thereby easily relieving the autoinhibited state of STX17(Fig. 2E).Then, we sought to understand how the STX17 SNARE motif

recognizes the mammalian ATG8 orthologs. We choseGABARAP and LC3A as two representatives of the GABARAPsubfamily and the LC3 subfamily, respectively, and carefullycharacterized their interactions with STX17(142–228). NMR-based analyses showed that both GABARAP and LC3A caninteract with STX17(142–228) (Fig. 3A and SI Appendix, Fig.S13A), and the major binding sites on STX17 are located withinthe N-terminal part (residues 170 to 191) of the STX17 Qa-SNARE motif (Fig. 3B and SI Appendix, Fig. S13B). Notably,the binding sites of the STX17 SNARE motif for interacting withmammalian ATG8 orthologs and the STX17 Habc domain arehighly overlapped (Figs. 1 B and C and 3 A and B and SI Ap-pendix, Fig. S13 A and B), therefore mechanistically explainingwhy mammalian ATG8 orthologs can compete with theN-terminal Habc domain for binding to the SNARE motif ofSTX17. Interestingly, detailed sequence analysis showed that theSTX17 SNARE region contains two putative LIR motifs,“WETL” (residues 172 to 175) and “FSLL” (residues 189 to192), although the critical aromatic Phe residue in the secondputative LIR motif is not strictly conserved in mammals (SIAppendix, Fig. S1A). Further NMR-based analyses revealed thatthe NMR resonances of the first putative LIR motif show muchmore significant changes than those of the second putative LIRregion when titrated with GABARAP or LC3A (Fig. 3 A and Band SI Appendix, Fig. S13 A and B), suggesting that only the firstputative LIR motif may directly participate in the interaction withGABARAP or LC3A. To further test whether these two putativeLIR motifs are directly involved in the GABARAP or LC3A bind-ing, we mutated the two crucial aromatic residues (W172 and F189)within the two putative LIR motifs of STX17(142–228) and con-structed three mutants, STX17(142–228) W172Q, STX17(142–228)F189Q, and the STX17(142–228) W172Q/F189Q double mutant.Then, we used these three mutants together with the wild-typeSTX17(142–228) and quantitatively compared their interactionswith GABARAP and LC3A using ITC-based assays (Fig. 3 C–F andSI Appendix, Fig. S13 C–F). The obtained ITC results showed thatthe W172Q mutation dramatically reduces and totally abolishes theinteraction of STX17(142–228) with GABARAP and LC3A, re-spectively (Fig. 3 C and D and SI Appendix, Fig. S13 C and D), whilethe F189Q mutation does not affect the binding of STX17(142–228)to GABARAP and LC3A (Fig. 3 C and E and SI Appendix, Fig.S13 C and E), confirming that only the first putative LIR motif ofSTX17 is directly involved in the interactions with GABARAP andLC3A (Fig. 3 A and B and SI Appendix, Fig. S13 A and B). Sur-prisingly, the STX17(142–228) W172Q and W172Q/F189Q mutantsstill displayed some residual binding abilities to GABARAP but notLC3A (Fig. 3 D and F and SI Appendix, Fig. S13 D and F), and theycan weakly bind to GABARAP with similar Kd values, ∼19 and ∼21μM, respectively (Fig. 3 D and F), implying that, except for the ca-nonical hydrophobic LIR core sequence, there are additional struc-tural features that may contribute to the interaction of STX17 withGABARAP but not LC3A. Finally, using analytical ultracentrifuga-tion analyses, we further elucidated that STX17(142–228) andGABARAP both form monomers in solution and, importantly, theycan interact with each other to form a 1:1 stoichiometric complex(Fig. 3G), consistent with our notion that the Qa-SNARE region ofSTX17 only contains one LIR motif.

The Overall Structure of the STX17 LIR Motif in Complex withGABARAP. To further elucidate the molecular mechanism gov-erning the interaction between the STX17 LIR motif and

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mammalian ATG8 proteins, we sought to determine their com-plex structures. Initially, we purified the STX17(142–228)–GABARAP complex to conduct a crystal screening but, unfor-tunately, we only obtained crystals with poor diffractions. Giventhat the STX17(142–228) fragment is basically unstructured, wefurther narrowed down the N- and C-terminal boundaries ofSTX17 and chose a STX17(167–188) fragment, which includesthe entire proven LIR motif but lacks the second putative LIRsequences (SI Appendix, Fig. S1A). Fortunately, using the puri-fied STX17(167–188)–GABARAP complex, we obtained goodcrystals that diffracted to 2.0-Å resolution. The crystal structure ofthe STX17 LIR–GABARAP complex was determined using themolecular replacement method (SI Appendix, Table S1). In the finalrefined structural model, each asymmetric unit contains fourSTX17 LIR–GABARAP complex molecules, and each STX17LIR–GABARAP complex has a 1:1 stoichiometry (Fig. 4A), in linewith our biochemical result (Fig. 3G). As expected, the GABARAPmolecule in the complex structure adopts a typical ATG8 homologprotein architecture consisting of two N-terminal α-helices followedby a ubiquitin-like core that is assembled by a four-stranded β-sheettogether with two α-helices (Fig. 4A). In the complex structure, theGABARAP-bound STX17 LIR motif is mainly composed of twoparts: an N-terminal extended structure formed by the canonicalLIR core containing the signature ΦXXΨ (WETL) sequences andan additional C-terminal 310-helix extension (Fig. 4 A and B and SIAppendix, Fig. S1A), in agreement with our aforementioned ITCresults (Fig. 3 D and F). The entire STX17 LIR motif packs ex-tensively with a solvent-exposed elongated groove mainly formed bythe α1-, α2-, and α3-helices together with the β2-strand ofGABARAP, burying a total surface area of ∼778 Å2 (Fig. 4 A andB and SI Appendix, Fig. S14A). Intriguingly, structural comparisonsof the STX17 LIR–GABARAP complex with currently knowncomplexes of ATG8s bound with unconventional LIR motifs thatcontain C-terminal extensions, such as the FYCO1 LIR–LC3Acomplex (Protein Data Bank [PDB] ID code 5CX3) (42), AnkBLIR–GABARAP complex (PDB ID code 5YIR), and AnkGLIR–GABARAPL1 complex (PDB ID code 5YIP) (43), revealed

that the overall interaction modes of the extended LIR motifs fromSTX17, FYCO1, and AnkB/G toward ATG8 family proteins arevery similar but, strikingly, only the STX17 LIR includes aC-terminal extension with a 310 helix (SI Appendix, Fig. S14B).

The Molecular Interface of the STX17 LIR–GABARAP Complex. De-tailed structural analysis of the binding interface of the STX17LIR–GABARAP complex revealed that the binding betweenSTX17 LIR and GABARAP is mediated by extensive hydro-phobic contacts and polar interactions (Fig. 4 B and C and SIAppendix, Fig. S14C). In particular, the aromatic side chainof W172 of STX17 LIR occupies a hydrophobic pocket ofGABARAP mainly assembled by the hydrophobic side chains ofI21, P30, L50, and F104 as well as the aliphatic side chain of K48and, meanwhile, the side-chain group of STX17 W172 also formsa hydrogen bond with the side chain of E17 located at the α2-helix of GABARAP (Fig. 4 B and C). In parallel, the hydro-phobic side chains of L175, L179, and L182 from STX17 LIRpack against a hydrophobic patch that is situated at the β2/α3-groove and formed by the side chains of the Y49, V51, P52, L55,F60, F62, and L63 residues of GABARAP (Fig. 4 B and C).Moreover, the backbone oxygen of STX17 E170 forms a stronghydrogen bond with the side chain of the K48 residue located atthe β2-strand of GABARAP, and the backbone oxygen andamide group of STX17 E173 form two backbone hydrogen bondswith the K48 and L50 residues of GABARAP (Fig. 4C). In ad-dition, the STX17 LIR–GABARAP complex is further stabilizedby two charge–charge interaction networks, one of which is lo-cated at the N-terminal region of GABARAP and is assembledby the negatively charged STX17 E170 residue coupled with thepositively charged H9 and K47 residues of GABARAP, while theother is formed between the negatively charged E173, D178, andE181 residues of STX17 LIR and positively charged K66 andR67 residues of GABARAP (Fig. 4 B and C and SI Appendix,Fig. S14C). Notably, all these key interface residues of STX17and GABARAP are highly conserved during evolution (SI Ap-pendix, Fig. S1). Using ITC and coimmunoprecipitation (co-IP)

Fig. 2. GABARAP can directly bind to STX17 and relieve the autoinhibited conformation of STX17. (A and B) Analytical gel filtration chromatographyanalyses of the interactions between GABARAP and STX17(142–228) (A) or STX17(1–228) (B). (C and D) ITC-based measurements of the binding affinities ofGABARAP with STX17(142–228) (C) or STX17(1–228) (D). (E) Superposition plot of the 1H-15N HSQC spectra of STX17(1–123) (red), STX17(1–123) titrated withSTX17(142–228) at a molar ratio of 1:1 (green), and STX17(1–123) saturated with STX17(142–228) followed by adding GABARAP at a molar ratio of 1:1:1(blue). For clarity, the Insets show enlarged views of two selected regions of the overlaid 1H-15N HSQC spectra. DP, differential power measured by theITC machine.

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analyses, we further verified the interactions observed in thestructure of the STX17 LIR–GABARAP complex. Consistentwith our structural data, the ITC results showed that individualpoint mutations of the key interface residues either from STX17or GABARAP, such as the E170A, W172Q, D178R, and L179Qmutations of STX17(142–228) or the K47E, K48E, L55A, L63Q,K66E, and R67E mutations of GABARAP, all largely reduce orcompletely disrupt the interaction between STX17(142–228) andGABARAP (Fig. 4D and SI Appendix, Fig. S15). Importantly,consistent with our in vitro ITC results, further co-IP experi-ments revealed that point mutations of key interface residuesincluding the K48E and R67E mutations of GABARAP and theW172Q and D178R mutations of STX17 all significantly atten-uate or essentially abolish the interaction between full-lengthSTX17 and GABARAP in cotransfected cells (Fig. 4E). Nota-bly, further detailed structure-based sequence alignment andstructural comparison analyses showed that several key binding-interface residues are quite different among the six mammalianATG8 orthologs (SI Appendix, Fig. S16). For instance, the resi-dues corresponding to the bulky hydrophobic L55 and F62 inGABARAP, which are critical for the hydrophobic interactionwith the C-terminal 310 helix (SI Appendix, Fig. S16B), are arelatively smaller Val residue in LC3A and LC3B and a polar Lysor Ser residue in LC3s, respectively (SI Appendix, Fig. S16A); theresidue corresponding to the positively charged K47 inGABARAP is a neutral Thr residue in LC3s, and the residuecorresponding to the positively charged R66 in GABARAP is aneutral Ser residue in LC3C (SI Appendix, Fig. S16A). Consistentwith these sequence- and structure-based analyses, further ITC-

based assays demonstrated that the GABARAP K47T/L55V/F62K triple mutant has a comparable binding affinity forSTX17(142–228) to that of LC3A and, conversely, the V58L/K65F double mutant of LC3A has a much increased bindingability for STX17(142–228) (SI Appendix, Fig. S17). In all, theidentification of these nonconserved interface residues amongdifferent mammalian ATG8 orthologs not only provided amechanistic explanation for the selective binding of STX17 LIRtoward different mammalian ATG8 orthologs but also rationalizedour aforementioned biochemical results that the STX17(142–228)W172Q and W172Q/F189Q mutants can weakly bind toGABARAP but not to LC3A that lacks the crucial bulky hydro-phobic L55 and F62 residues for interacting with the C-terminal 310-helix extension of STX17 (Fig. 3 D and F and SI Appendix, Fig.S13 D and F).

The Biochemical and Structural Properties of the STX17–SNAP29–VAMP8SNARE Complex. Given that GABARAP can occupy the N-terminalpart of the STX17 SNARE motif and form a stable complex withSTX17, next we wanted to know whether GABARAP may affectthe assembly of the STX17–SNAP29–VAMP8 SNARE complex.Using analytical gel filtration chromatography-based analyses, weshowed that the Qb- and Qc-SNARE motifs of SNAP29 alone areunable to interact with or disturb the GABARAP–STX17 complex(SI Appendix, Fig. S18A); however, in the presence of an additionalVAMP8 R-SNARE motif, a stable SNARE complex containing theSTX17 Qa-SNARE motif, the VAMP8 R-SNARE motif, as well asthe Qb- and Qc-SNARE motifs of SNAP29, was readily formed (SIAppendix, Fig. S18 B and C), indicating that SNAP29 and VAMP8

Fig. 3. STX17 Qa-SNARE region contains one LIR motif that is required for interaction with GABARAP. (A) Superposition plot of the assigned 1H-15N HSQCspectra of STX17(142–228) titrated with increasing molar ratios of unlabeled GABARAP proteins. In this representation, the four core residues (F189, S190,L191, L192) of the second putative LIR motif of STX17 are further highlighted and colored in sky blue. (B) Plot of backbone amide chemical shift differencesand peak broadening as a function of the residue number of STX17(142–228) between the wild type and the protein titrated with GABARAP at a molar ratioof 2:1. In this representation, the residues with disappeared NMR peaks due to peak broadening are shown in black, and the combined 1H and 15N chemicalshift changes are defined as shown in Eq. 1. In addition, the corresponding four core residues (residues 189 to 192) of the second putative LIR motif of STX17are also indicated. (C–F) ITC-based measurements showing the binding affinities of GABARAP with STX17(142–228) (C), STX17(142–228) W172Q mutant (D),STX17(142–228) F189Q mutant (E), and STX17(142–228) W172Q/F189Q double mutant (F). (G) Overlay plot of the sedimentation velocity data ofSTX17(142–228) (black), GABARAP (red), and STX17(142–228)–GABARAP complex (blue). These results demonstrate that STX17(142–228) and GABARAP formmonomers and interact with each other to form a 1:1 stoichiometric complex.

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together can easily compete with GABARAP for binding to STX17to assemble the STX17–SNAP29–VAMP8 SNARE complex. Sincethe membrane fusion between the double-membraned autophago-some and single-membraned lysosome is morphologically distinctfrom the conventional fusion event between two single-membranedvesicles, we wondered whether this autophagic STX17–SNAP29–VAMP8 SNARE complex has striking biochemical properties. Totest this hypothesis, we first purified this autophagic SNAREcomplex, which includes the SNARE motifs of STX17 and VAMP8together with their short neck regions, and the Qb- and Qc-SNAREmotifs of SNAP29, and then used circular dichroism (CD) spec-troscopy to examine its secondary structure features and thermalstability. In contrast to that of the isolated SNARE regions ofSTX17 and VAMP8 as well as the full-length SNAP29, the SNAREcomplex showed significant characteristic α-helical content, as

indicated by its CD spectrum (Fig. 5A). It is noteworthy that theisolated SNARE region of STX17 is intrinsically disordered basedon the CD analysis (Fig. 5A), consistent with the aforementionedNMR results (SI Appendix, Fig. S2). Interestingly, further CD-basedanalysis revealed that with increasing temperature, this autophagicSNARE complex undergoes unfolding with a melting temperature(Tm) of ∼85 °C (Fig. 5B), which is a little higher than that of the lateendosomal SNARE complex (Tm 78 °C) but slightly lower than itsearly endosomal (Tm 87 °C) and neuronal (Tm 90 °C) counterparts(46–48). Subsequent reduction of the temperature of the sample ledto the initiation of refolding at a much lower temperature of ∼51 °Cand further cooling to 10 °C resulted in a partial refolding of theoriginal α-helical content (Fig. 5B), showing the characteristicunfolding–refolding hysteresis of a typical SNARE core complex(28, 49).

Fig. 4. Structural analyses of the STX17 LIR–GABARAP complex. (A) Ribbon diagram showing the overall structure of the STX17 LIR–GABARAP complex. Inthis drawing, GABARAP is shown in forest green, and the STX17 LIR motif is in magenta. (B) The combined surface representation and ribbon-stick modelshowing the molecular interface of GABARAP in the STX17 LIR–GABARAP complex. In this representation, GABARAP is shown in the surface model and STX17LIR is in the ribbon-stick model. The hydrophobic amino acid residues of GABARAP in the surface model are drawn in yellow, the positively charged residuesare in blue, the negatively charged residues are in red, and the uncharged polar residues are in gray. (C) Stereoview of the ribbon-stick representationshowing the detailed interactions between GABARAP and STX17 LIR. In this drawing, the side chains of the key residues are shown in stick-ball mode, and thehydrogen bonds involved in the binding are shown as dotted lines. (D) The measured binding affinities between various forms of GABARAP–LC3A and theSTX17 Qa-SNARE motif or their mutants by ITC-based analyses. (E) Mutagenesis-based co-IP assays confirming the interactions between GABARAP and STX17observed in the determined STX17 LIR–GABARAP complex structure. IB, immunoblotting.

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We also solved the crystal structure of this autophagic SNAREcore complex (SI Appendix, Table S1). As expected, this auto-phagic SNARE complex forms a four-helix bundle with all fourhelices aligned in parallel, and STX17 and VAMP8 each con-tribute one helix while SNAP29 contributes two helices to the

bundle (Fig. 5C and SI Appendix, Fig. S19). Notably, a similaroverall architecture was also observed in a previously reportedstructure of the STX17–SNAP29–VAMP8 SNARE complexwith slightly different protein boundaries (16). Interestingly, theelectrostatic potential surface of this autophagic SNARE complex

Fig. 5. Biochemical and structural characterizations of the STX17–SNAP29–VAMP8 SNARE complex. (A) Overlay plot of the CD spectra of the STX17–SNAP29–VAMP8 SNARE complex and related individual autophagic SNARE proteins. (B) Thermal unfolding and refolding analyses of the STX17–SNAP29–VAMP8 SNARE complex monitored by CD spectroscopy at 222 nm. (C) Ribbon diagram showing the overall structure of the STX17–SNAP29–VAMP8 SNAREcomplex. (D) Surface charge potential representation (contoured at ±5 kT/eV; blue/red) of the STX17–SNAP29–VAMP8 SNARE complex with the same ori-entation as in C, revealing two highly negatively charged patches. (E) Detailed interior interactions of the autophagic STX17–SNAP29–VAMP8 SNARE complexformed by 16 layers of interacting amino acid side chains.

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showed two highly negatively charged patches (Fig. 5D), whichare on opposite sides of the surface and are implicated in thebinding to the NSF-mediated SNARE-disassembly machinery(28, 50). Similar to other known SNARE complexes, the interiorof this autophagic SNARE complex is formed by 16 layers ofinteracting amino acid side chains that are mostly hydrophobic,and the hydrophilic 0 layer is formed by three Gln residues (Q196of STX17 and Q84 and Q230 of SNAP29) and one Arg resi-due (R37 of VAMP8) (Fig. 5E and SI Appendix, Fig. S19). De-tailed structural analyses of the other 15 layers revealed thatthe −fifth, −first, first, fourth, and eighth layers are all formed byfour hydrophobic side chains, whereas the −seventh, −sixth, −fourth,second, third, fifth, and seventh layers are all composed of threehydrophobic side chains and one polar side chain of a Ser or Thrresidue, of which the hydroxyl group throughout points to the out-side of the layer (Fig. 5E). Notably, the arrangements of the sidechains in the −third, −second, and sixth layers are highly asymmetric(Fig. 5E). Next, we sought to use the glutathione S-transferase(GST)–fusion protein pull-down assay to verify the contributionsof different intact layers to the assembly of the autophagic SNAREcomplex. We individually disrupted the intact −seventh, −fifth, 0,fourth, and seventh layers of the SNARE complex by mutation ofkey residues involved in the layer formation either from STX17 orSNAP29. The W172Q (−seventh layer), L179Q (−fifth layer), andQ196L (0 layer) mutations of STX17 as well as the L213Q (−fifthlayer) and Q230L (0 layer) mutations of SNAP29 essentially abol-ished the SNARE core complex formation, whereas the A210Q(fourth layer) and L221Q (seventh layer) mutations of STX17 onlypartially weakened the core complex formation (Fig. 5E and SIAppendix, Fig. S20), suggesting that the N-terminal and central layersare much more important than the C-terminal layers during theassembly of the autophagic SNARE complex, in keeping with thezipper-like mode for canonical SNARE complex assembly (28, 51).In addition, the four-helix bundle structure of this autophagicSNARE complex is further stabilized by extensive surface interac-tions between different helices (SI Appendix, Figs. S17 and S21).

DiscussionIn this study, we uncovered that the STX17 Qa-SNARE motifcan directly interact with its N-terminal Habc domain, suggestingthat, in isolation, STX17 adopts a closed conformation. Con-sidering the poor quality of the 1H-15N HSQC spectrum of theSTX17 Habc region in the presence of the STX17 SNARE motif(Fig. 1D and SI Appendix, Fig. S3), we sought to use X-raycrystallography to solve the autoinhibited structure of STX17Habc in complex with the Qa-SNARE motif. Unfortunately,after numerous trials, we failed to obtain good crystals forstructure determination, presumably due to the dynamic natureof this unstable complex, as indicated by our NMR analyses(Fig. 1 B and D). Hence, further studies are required to elucidatethe detailed molecular mechanism underlying the interactionbetween the Habc domain and the Qa-SNARE motif of STX17.Strikingly, a similar intramolecular interaction between the

N-terminal Habc domain and the C-terminal Qa-SNARE motifwas observed in Syntaxin1, a neuronal Qa-SNARE protein in-volved in synaptic membrane fusion (52). However, unlike thatof Syntaxin1, the binding of the STX17 SNARE motif to its Habcdomain can induce a partial unfolding of the extreme C-terminalα-helix of the STX17 Habc domain (Fig. 1D and SI Appendix,Fig. S4C). Importantly, previous studies of Syntaxin1 had well-demonstrated that the closed conformation of Syntaxin1 is es-sential for its interaction with Munc18-1, a major regulator ofsynaptic vesicle fusion, and represents a critical intermediate forneuronal exocytosis (52). Similarly, STX17 was reported to in-teract with the HOPS tethering complex through the Munc18-like subunit Vps33 for mediating autophagosome–lysosome fu-sion (15, 17, 53). However, whether HOPS binds to a closed oran open conformation of STX17 during the autophagosome–

lysosome fusion process and what the potential in vivo function isof the conformational change of STX17 Habc induced by theQa-SNARE motif remain to be elucidated.Our study showed that SNAP29 alone is unable to open the

closed conformation of STX17 to form a stable STX17–SNAP29(Qa/Qbc) t-SNARE complex in vitro (SI Appendix, Figs. S6Aand S7B). However, mammalian ATG8 orthologs, such asGABARAP, could easily release the autoinhibited conformationof STX17 by competitively binding to the N-terminal region ofthe STX17 SNARE motif and form a stable binary complex withSTX17. Since previous relevant functional studies have well-demonstrated that the inactivation of all six mammalian ATG8orthologs or the mammalian ATG8 conjugation machinery sig-nificantly attenuates the recruitment of STX17 to the autopha-gosome as well as the fusion between the autophagosome andlysosome (19, 20), our work may provide direct structural evi-dence for the essential function of mammalian ATG8s in me-diating the autophagosome–lysosome fusion process. However,given that STX17 is exclusively recruited to the external mem-brane of the autophagosome instead of the phagophore that isalso decorated with mammalian ATG8s (13, 20), the sole in-teractions between STX17 and mammalian ATG8s are likelyinsufficient to accomplish the recruitment of STX17 to theemerging autophagosome rather than the phagophore. Inter-estingly, a recent study showed that the autophagy-related smallGTPase IRGM in its active form can directly associate withSTX17 by binding to its two transmembrane domains (30), whichwas proven to be essential for the translocation of STX17 toautophagic membranes (13). Unfortunately, we were unable toobtain soluble IRGM proteins either from Escherichia coli orinsect cells, thereby preventing detailed biochemical and struc-tural characterizations. Additional work is required to elucidatethe detailed mechanism governing the temporal and spatialregulation of the interaction of STX17 with mammalian ATG8sas well as the recruitment of STX17 to the autophagosome.Furthermore, our systemic biochemical and structural char-

acterizations revealed that the key binding-interface residues ofSTX17 involved in GABARAP binding and the STX17–SNAP29–VAMP8 SNARE complex assembly are heavily over-lapped (Figs. 4C and 5E and SI Appendix, Figs. S1A and S19B),such as the W172 and L175 residues of STX17. Therefore, due tothe potential steric exclusion, once the STX17–SNAP29–VAMP8 SNARE complex is formed, GABARAP and likelyother ATG8 orthologs are unable to associate with this auto-phagic SNARE complex (SI Appendix, Fig. S18 B and C). In-terestingly, based on careful structural analyses (Fig. 4C and SIAppendix, Fig. S22A), we rationally designed and obtained aSTX17 D178R point mutation, which disrupted the interactionof STX17 with GABARAP without affecting the assembly andthe stability of the STX17–SNAP29–VAMP8 SNARE complex(SI Appendix, Figs. S15B and S22B). Therefore, this mutationmay potentially be useful for the future functional study ofSTX17 as well as the dissection of different roles mediated by theSTX17–ATG8 ortholog interaction and this STX17-containingSNARE complex for autophagosome–lysosome fusion.In summary, we proposed a model depicting three different

states of STX17 in cooperation with mammalian ATG8s as wellas the autophagy-related SNARE proteins SNAP29 and VAMP8during the autophagosome–lysosome fusion process (Fig. 6). Inthis model, STX17 alone is in an autoinhibited closed state and,particularly, its Habc region somehow packs with the N-terminalpart of its Qa-SNARE motif to stabilize the intrinsically disor-dered SNARE motif of STX17, thereby preventing its unnecessarydegradation or interactions with other proteins on the autopha-gosome before assembling the STX17–SNAP29–VAMP8 SNAREcomplex (Fig. 6). However, in the presence of GABARAP/LC3family proteins, the mammalian ATG8 ortholog can competitivelybind to the N-terminal part of the STX17 Qa-SNARE motif that

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contains an extended LIR motif and abolish the autoinhibitedconformation of STX17 (Fig. 6), thereby releasing the N-terminalHabc domain of STX17 and inducing its conformational rear-rangement. Then, relevant tethering factors, such as the HOPScomplex, ATG14, and EPG5, are recruited, which in turn workwith STX17 and promote the further recruitment of cytosolic Qbc-SNARE SNAP29 and bring the VAMP8-residing lysosome intoclose proximity to the autophagosome (Fig. 6). Finally, mediatedby their respective SNARE motifs, STX17, SNAP29, and VAMP8assemble into the autophagic SNARE complex to clamp themembranes together and initiate the fusion between the auto-phagosome and lysosome, eventually leading to the formation ofthe autolysosome and the subsequent degradation of the enclosedmaterials (Fig. 6).

Materials and MethodsProtein Expression and Purification. Different DNA fragments encoding hu-man STX17, SNAP29, VAMP8, and ATG8s and other related DNA fragmentswere amplified by PCR from the full-length human complementary DNA(cDNA). All these fragments were cloned into in-house modified versions ofthe pET32a vector for recombinant protein expression. For the coimmuno-precipitation assay, full-length STX17 and GABARAP DNA fragments werecloned into pmCherry-C1 and pEGFP-C1 vectors, respectively. All of the pointmutations of STX17, SNAP29, GABARAP, and LC3A used in this study werecreated using the standard PCR-based mutagenesis method, further checkedby PCR screen using 2× Taq Master Mix (Vazyme Biotech) enzyme andconfirmed by DNA sequencing.

Recombinant proteins were expressed in BL21 (DE3) E. coli cells induced by100 μM isopropyl β-D-1-thiogalactopyranoside at 16 °C. The bacterial cellpellets were resuspended in binding buffer (50 mM Tris, pH 7.9, 500 mMNaCl, 5 mM imidazole), and then lysed by the FB-110XNANO homogenizermachine (Shanghai Litu Machinery Equipment Engineering). Then the lysatewas centrifuged at 35,000 × g for 30 min to remove debris. His6-taggedproteins were purified using Ni2+-NTA agarose (GE Healthcare) affinitychromatography followed by size-exclusion chromatography (Superdex 75or 200 column; GE Healthcare) in a buffer containing 20 mM Tris·HCl (pH7.5), 100 mM NaCl, and 1 mM dithiothreitol (DTT). Purities and molecularmasses were verified by sodium dodecyl sulfate/polyacrylamide gel electro-phoresis (SDS/PAGE) analysis.

To obtain the STX17 LIR–GABARAP complex proteins used for crystalli-zation, GST-STX17(167–188) and His-GABARAP were first copurified by glu-tathione Sepharose 4B (GE Healthcare) affinity chromatography. Then, theN-terminal tags were cleaved by 3C protease, and the GST tag was furtherremoved by glutathione Sepharose 4B affinity chromatography. Finally, theSTX17(167–188)–GABARAP complex was further purified on a Superdex75 size-exclusion column that was equilibrated with a column buffer con-taining 20 mM Tris·HCl (pH 7.5), 100 mM NaCl, and 1 mM DTT. For theSTX17–SNAP29–VAMP8 SNARE core complex proteins used for crystalliza-tion, Trx-STX17(142–228), MBP-SNAP29(40–126), GB1-SNAP29(191–258), andTrx-VAMP8(8–75) were first copurified by Ni2+-NTA agarose (GE Healthcare)affinity chromatography followed by size-exclusion chromatography(Superdex 200 column). Fractions containing the SNARE complex were col-lected and digested by 3C protease, loaded onto a MonoQ 10/10 ion-exchange column (GE Healthcare), and eluted with a linear NaCl gradientup to 500 mM to remove N-terminal tags. Finally, the autophagic STX17–

SNAP29–VAMP8 SNARE complex was further purified using a Superdex75 size-exclusion column equilibrated with a column buffer containing 20 mMTris·HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 1 mM ethylenediaminetetraaceticacid (EDTA).

Uniformly 15N- or 15N/13C-labeled proteins were prepared by growingbacteria in M9 minimal medium using 15NH4Cl (Cambridge Isotope Labora-tories; NLM-467) as the sole nitrogen source or 15NH4Cl and [13C6]glucose(Cambridge Isotope Laboratories; CLM-1396) as the sole nitrogen and carbonsources, respectively.

NMR Spectroscopy. The stable isotope-labeled protein samples for NMRstudies were concentrated to ∼0.1 mM for titration experiments and∼0.6 mM for backbone resonance assignment experiments in 50 mM po-tassium phosphate buffer containing 50 mM NaCl (pH 6.5) and 1 mM DTT.NMR spectra were acquired at 25 °C on an Agilent 800-MHz spectrometerequipped with an actively z gradient-shielded triple-resonance cryogenicprobe. Backbone resonance assignments of STX17(142–228) and theSTX17 SNARE-bound Habc domain were achieved using a suite of hetero-nuclear correlation experiments including HNCO, HNCA, CA(CO)NH,HNCACB, and CBCA(CO)NH using a 15N/13C-labeled protein sample togetherwith a three-dimensional 15N-separated NOESY (54).

Analytical Gel Filtration Chromatography. Analytical gel filtration chroma-tography was carried out on an AKTA FPLC System (GE Healthcare). Proteinsamples were loaded onto a Superose 12 10/300 GL column (GE Healthcare)equilibrated with a buffer containing 20 mM Tris·HCl (pH 7.5), 100 mM NaCl,and 1 mM DTT.

Isothermal Titration Calorimetry Assay. ITC measurements were carried out ona MicroCal PEAQ-ITC calorimeter or an automated system (Malvern) at 25 °C.For each ITC experiment in this study, the protein samples in the cell and inthe syringe were exchanged into the same buffer condition containing20 mM Tris (pH 7.5), 100 mM NaCl, and 1 mM DTT using a HiPrep 26/10desalting column. The titration processes were performed by injecting 40-μLaliquots of the syringe sample (∼500 μM) into the cell sample (∼50 μM) attime intervals of 2 min to ensure that the titration peak returned to base-line. In addition, relevant reference control experiments, in which the pro-tein samples in the syringe are titrated into the control buffers, were alsoconducted. For each ITC dataset, the reference data would be subtractedfrom the raw data to obtain the net result for the final fitting analysis. Thetitration data were analyzed using the Malvern MicroCal PEAQ-ITC analysisprogram. The Kd error is the fitted error obtained from the data analysissoftware when using the one-site binding model to fit the ITC data.

Analytical Ultracentrifugation. Sedimentation velocity experiments wereperformed on a Beckman XL-I analytical ultracentrifuge equipped with aneight-cell rotor at 42,000 rpm at 20 °C. The partial specific volume of dif-ferent protein samples and the buffer density were calculated using theprogram SEDNTERP (http://www.rasmb.org/). The final sedimentation ve-locity data were analyzed and fitted to a continuous sedimentation coeffi-cient distribution model using the program SEDFIT (55). The fitting resultswere further output to Origin 9.0 software and aligned with each other.

Protein Crystallization and Structural Elucidation. Crystals of the STX17 LIR–GABARAP complex and autophagic STX17–SNAP29–VAMP8 SNARE complexwere obtained using the sitting-drop vapor-diffusion method at 16 °C.

Fig. 6. Proposed cartoon model illustrating the three different states as well as the potential working mode of STX17 in cooperation with mammalianATG8s, autophagic SNARE proteins SNAP29 and VAMP8, and relevant tethering factors for facilitating the membrane fusion process between the auto-phagosome and lysosome in macroautophagy.

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Specifically, crystals of the STX17 LIR–GABARAP complex were formed about1 wk after the freshly purified STX17 LIR–GABARAP complex (10 mg/mL in20 mM Tris·HCl, pH 7.5, 100 mM NaCl, 1 mM DTT) was mixed with an equalvolume of reservoir solution containing 0.12 M alcohols, 50% (vol/vol) pre-cipitant Mix 4, and buffer system 3 at pH 8.5 from the Morpheus Screen Kit(Molecular Dimensions). Meanwhile, crystals of the STX17–SNAP29–VAMP8SNARE complex (20 mg/mL in 20 mM Tris·HCl, pH 7.5, 100 mM NaCl, 1 mMDTT, 1 mM EDTA) were grown from buffer containing 2.5 M ammoniumnitrate (pH 4.6) and 0.1 M sodium acetate trihydrate. Before the diffractionexperiments, glycerol as appropriate was added as the cryoprotectant. X-raydatasets were collected at beamline BL17U1 or BL19U1 of the ShanghaiSynchrotron Radiation Facility (SSRF) (56). The diffraction data were pro-cessed and scaled using HKL2000 (57).

The phase problem of the STX17 LIR–GABARAP complex was solved by themolecular replacement method using the modified structure of GABARAP(PDB ID code 5YIR) with PHASER (58). Meanwhile, the phase problems of theSTX17–SNAP29–VAMP8 SNARE complex were solved by the molecular re-placement method using the crystal structure of the endosomal SNARE corecomplex (PDB ID code 1GL2) as the search model. All initial structural modelswere rebuilt manually using Coot (59) and then refined using REFMAC (60)or PHENIX (61). The qualities of the final model were validated by MolPro-bity (62). The final refinement statistics of the solved structures in this studyare listed in SI Appendix, Table S1. All of the structure figures were preparedusing the program PyMOL (https://pymol.org/2/).

Coimmunoprecipitation Assay. HEK293T cells transiently expressing proteinswere harvested, washed with phosphate-buffered saline (PBS) buffer, andlysed for 1 h at 4 °C in lysis buffer containing 50 mM Tris·HCl (pH 7.8), 50 mMNaCl, 0.4% Nonidet P-40, 0.5 mM phenylmethanesulfonyl fluoride, andprotease inhibitor mixture (AMRESCO). Lysates were centrifuged and thensupernatants were incubated with the appropriate antibody pretreated withrProtein G agarose (Invitrogen) for 3 h with rotation at 4 °C. Precipitatedproteins were washed with lysis buffer five times and then collected by briefcentrifugation. Subsequently, the precipitated proteins were resolved bySDS/PAGE and detected by immunoblotting using a chemical luminescence-based detection method.

Circular Dichroism Spectroscopy. CD measurements were performed using aChirascan instrument (Applied Photophysics). A Hellma quartz cuvette with a

path length of 0.5 mmwas used. CD spectra were obtained by measuring thepurified wild type or mutants of the autophagic STX17–SNAP29–VAMP8SNARE core complex at a concentration of 20 μM in a buffer containing40 mM sodium phosphate buffer (pH 6.5). For the thermal melting experi-ment, the wavelength was set to 222 nm and the temperature from 10 to105 °C. Then the unfolding measurement was started by gradually increas-ing the temperature to 105 °C at a rate of 30 °C/h. Subsequently, the tem-perature for refolding was again lowered to 10 °C at a rate of 30 °C/h.

GST Pull-Down Assay. Direct interactions between different Syntaxin17,SNAP29, and VAMP8 proteins were assayed in PBS (pH 7.4). The Syntaxin17fragment (residues 142 to 225/228) was tagged with GST, while the SNAP29fragment (residues 40 to 126), SNAP29 fragment (residues 191 to 258), andVAMP8 fragment (residues 8 to 66/71) were tagged with MBP-His6, GB1-His6,and Trx-His6, respectively. Fifty micrograms of GST-tagged proteins and His-tagged proteins was mixed at a molar ratio of 1:2 in 1 mL of the assay buffer.The GST-STX17–SNAP29–VAMP8 complexes were pelleted by adding 30 μLfresh glutathione Sepharose 4B (GE Healthcare) beads. The pellets werewashed six times with 1 mL of the assay buffer, and subsequently elutedwith 60 μL of 30 mM glutathione buffer. Ten microliters of each elutedsample was separated by 12% SDS/PAGE and analyzed using Coomassie bluestaining or Western blot.

Data Availability. The coordinates and structure factors of the STX17 LIR–GABARAP complex and the STX17–SNAP29–VAMP8 SNARE complex repor-ted in this paper have been deposited in the Protein Data Bank (PDB IDcodes 7BV4 and 7BV6, respectively).

ACKNOWLEDGMENTS. We thank SSRF BL17U1 and BL19U1 for X-raybeamtime, Dr. Jianchao Li for help with X-ray diffraction data collection,and Prof. Jiahuai Han for the full-length Syntaxin17, SNAP29, and VAMP8cDNA. This work was supported by grants from the National Key R&DProgram of China (2016YFA0501903), National Natural Science Foundationof China (31470749, 21621002, 91753113, 21822705), Science and Technol-ogy Commission of Shanghai Municipality (17JC1405200), Strategic PriorityResearch Program of the Chinese Academy of Sciences (XDB20000000), andthe start-up fund from the State Key Laboratory of Bioorganic and NaturalProducts Chemistry and Chinese Academy of Sciences.

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