Decoding three distinct states of the Syntaxin17 SNARE motif in mediating –lysosome fusion

Ying 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 of Sciences, 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–sensitive RILP, TECPR1, PLEKHM1, BRUCE, and Pacer (22–27). factor attachment receptor (SNARE) protein, can associate However, the detailed molecular mechanisms underlying the co- with ATG8 family SNAP29 and VAMP8 to facilitate the operation of these proteins to promote the formation of the membrane fusion process between the double-membraned auto- autolysosome are still not well-understood. phagosome and single-membraned lysosome in mammalian mac- Cellular membrane fusion processes are known to be medi- roautophagy. However, the inherent properties of Syntaxin17 and ated by SNARE proteins, which can assemble into a membrane- the mechanistic basis underlying the interactions of Syntaxin17 bridging four-helix bundle (composed of Qa-, Qb-, Qc-, and with its binding proteins remain largely unknown. Here, using R-SNAREs) to provide the mechanical thrust for effectively biochemical, NMR, and structural approaches, we systemically driving membrane fusion (28). The fusion event between the characterized Syntaxin17 as well as its interactions with ATG8 double-membraned autophagosome and the single-membraned family proteins, SNAP29 and VAMP8. We discovered that Syn- lysosome during in mammals is reported to be me- taxin17 alone adopts an autoinhibited conformation mediated diated by two autophagy-specific SNARE complexes, the Syn- by a direct interaction between its Habc domain and the Qa- taxin17 (hereafter called STX17)–SNAP29–VAMP8 SNARE SNARE motif. In addition, we revealed that the Qa-SNARE region complex and the recently discovered YKT6–SNAP29–Syntaxin7 of Syntaxin17 contains one LC3-interacting region (LIR) motif, SNARE complex (13, 14). As the key player for promoting

which preferentially binds to GABARAP subfamily members. Im- autophagosome–lysosome fusion, the STX17-containing SNARE BIOPHYSICS AND COMPUTATIONAL BIOLOGY portantly, the GABARAP binding of Syntaxin17 can release its complex is composed of the autophagosomal SNARE STX17 autoinhibited state. The determined crystal structure of the Syn- (Qa-SNARE), the cytosolic SNARE SNAP29 (Qbc-SNAREs), and taxin17 LIR–GABARAP complex not only provides mechanistic in- the lysosomal SNARE VAMP8 (R-SNARE) (Fig. 1A). Structurally, sights into the interaction between Syntaxin17 and GABARAP but SNAP29 mainly contains Qb- and Qc-SNARE motifs, while also reveals an unconventional LIR motif with a C-terminally ex- VAMP8 is composed of an R-SNARE motif followed by a trans- tended 310 helix for selectively binding to ATG8 family proteins. membrane domain (Fig. 1A). As a Qa-type SNARE protein, STX17 Finally, we also elucidated structural arrangements of the autopha- contains an N-terminal Habc domain, a Qa-SNARE motif followed gic Syntaxin17–SNAP29–VAMP8 SNARE core complex, and uncov- by two unique tandem transmembrane domains (Fig. 1A). The two ered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct Significance states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome–lysosome fusion process. Macroautophagy is essential for the maintenance of cellular homeostasis and physiology in mammals, and relies on vesicle autophagy | SNARE | Syntaxin17 | GABARAP | fusion between the autophagosome and the lysosome, forming – autophagosome lysosome fusion the autolysosome to degrade unwanted cytosolic contents for recycling. The membrane fusion between the autophagosome acroautophagy (hereafter referred to as autophagy) relies and lysosome requires ATG8 family proteins and autophagy- Mon the double-membraned vesicle called the autophago- related SNARE proteins including Syntaxin17, VAMP8, and some to fuse with the lysosome, forming the autolysosome for SNAP29, but with poorly understood mechanisms. In this study, degradation of enclosed cytoplasmic materials in through systemic biochemical and structural characterizations, (1–5). Through autophagy, eukaryotic cells can recycle macro- we reveal three different states of the key autophagosomal molecular constituents, such as bulk protein aggregates, glycogen, SNARE protein Syntaxin17 and provide mechanistic insights into dysfunctional organelles, and invading pathogens, to maintain the autoinhibited state of Syntaxin17 as well as its interactions cellular homeostasis and/or adapt to multiple cellular stresses (1, with ATG8 family proteins, SNAP29 and VAMP8. Our findings 2). Thereby, autophagy plays critical roles in numerous physio- are valuable for further understanding the functions of Syn- logical processes, such as energy metabolism, immune response, taxin17 in the autophagosome–lysosome fusion process. embryogenesis, and aging (6–8). Dysfunctions of autophagy are associated with many human diseases, including cancer, immune Author contributions: Y.L. and L.P. designed research; Y.L., X.C., and M.L. performed – research; Yingli Wang contributed new reagents/analytic tools; Y.L., X.C., M.L., Yingli disorders, and neurodegenerative diseases (8 11). During the Wang, T.F., Z.Z., Yaru Wang, X.G., X.X., J.L., and L.P. analyzed data; and Y.L., X.C., and autophagy pathway, the formation of the autolysosome represents L.P. wrote the paper. one of the essential steps for ultimate autophagic degradation, and The authors declare no competing interest. depends on the tight coordination of autophagic vesicle fusions (1, This article is a PNAS Direct Submission. Y.Y. is a guest editor invited by the 4–6, 12). So far, many proteins have been identified as being in- Editorial Board. volved in these processes in mammals, including autophagic Published under the PNAS license. N-ethylmaleimide–sensitive (NSF) factor attachment protein re- 1To whom correspondence may be addressed. Email: [email protected]. ceptor (SNARE) proteins (13, 14), relevant tethering factors such This article contains supporting information online at https://www.pnas.org/lookup/suppl/ as the HOPS complex, ATG14, and EPG5 (15–18), ATG8 family doi:10.1073/pnas.2006997117/-/DCSupplemental. proteins (19–21), and related regulatory proteins including Rab7,

www.pnas.org/cgi/doi/10.1073/pnas.2006997117 PNAS Latest Articles | 1of12 Downloaded by guest on October 1, 2021 Fig. 1. NMR-based characterizations of the interaction between the STX17 Qa-SNARE motif and its N-terminal Habc domain. (A) A schematic diagram showing 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 STX17 and 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-labeled STX17(142–228) titrated with increasing molar ratios of unlabeled STX17(1–123). (C) Plot of backbone amide chemical shift differences and peak broadening as 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 this representation, the residues with disappeared NMR peaks due to peak broadening are shown in black and the combined 1Hand15N chemical shift changes are defined as

1=2 2 2 Δppm = []()ΔδHN + ()ΔδN × αN , [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) used to 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 increasing molar 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, parts per million.

2of12 | www.pnas.org/cgi/doi/10.1073/pnas.2006997117 Li et al. Downloaded by guest on October 1, 2021 transmembrane domains of STX17 are demonstrated to form a interactions with the ATG8 family proteins, SNAP29 and hairpin structure and are required for the localization of STX17 on VAMP8, and uncovered three different states of STX17. Spe- the autophagosome (13). The Qa-SNARE motif of STX17 can cifically, we discovered that the isolated STX17 adopts an coassemble with SNAP29 Qb-SNARE, Qc-SNARE motifs, and the autoinhibited “closed” conformation, in which the N-terminal R-SNARE motif of VAMP8, forming the SNARE core complex half of the STX17 Qa-SNARE motif occupies the Habc do- (13), and the structure of this core SNARE complex was deter- main of STX17. In addition, we revealed that STX17 only con- mined in a previous study from Zhong’s group (16). However, the tains one LIR motif, which preferentially binds to GABARAP biochemical properties of this autophagic SNARE complex and the subfamily members. We determined the high-resolution struc- detailed molecular basis underpinning the regulation of this auto- ture of the STX17 LIR–GABARAP complex, and uncovered the phagic SNARE complex formation are still largely unknown. In- molecular mechanism underpinning the interaction between triguingly, in addition to its canonical role in assembling the STX17 and GABARAP. Notably, the determined STX17 LIR– SNARE core complex, the Qa-SNARE motif of STX17 is also GABARAP complex structure also highlights the importance of implicated in interactions with many other autophagy-related pro- the C-terminal extension following the LIR core motif for some teins, such as ATG14 (16), TBK1 (29), and ATG8 family proteins LIR-containing proteins to selectively interact with ATG8 family (30). In particular, a recent study showed that STX17 can directly proteins. Finally, we also investigated the biochemical and interact with ATG8 family proteins and IRGM, an autophagy- structural features of the autophagic STX17–SNAP29–VAMP8 related small GTPase, through its Qa-SNARE motif region and SNARE core complex, and elucidated characteristic biochemical two transmembrane domains, respectively (30). Importantly, these properties and structural arrangements of this autophagic SNARE interactions of STX17 with ATG8 family proteins and IRGM are complex. essential for the efficient recruitment of STX17 to the autophago- some (30). However, due to the lack of detailed structural charac- Results terizations, the molecular mechanisms governing the interactions of The STX17 SNARE Motif Can Interact with the N-Terminal Habc STX17 with these proteins remain elusive. Domain of STX17 and Alter Its Conformation. To gain molecular ATG8 family proteins are small -like proteins, and include insights into the function of STX17 in the autophagosome– six orthologs in mammals known as MAP1LC3A (LC3A), lysosome fusion process, we first conducted a detailed sequence MAP1LC3B (LC3B), MAP1LC3C (LC3C), GABARAP, GABAR- alignment analysis of the cytoplasmic region of STX17 and found APL1, and GABARAPL2 (31–33). They can be further classified that the N-terminal Habc region (residues 1 to 123) and the Qa- into two subfamilies, the LC3 subfamily and the GABARAP sub- SNARE motif (residues 167 to 224) of STX17 are highly con- SI Appendix A family (31, 32). are decorated on the membrane of the served during evolution ( , Fig. S1 ), in line with their BIOPHYSICS AND COMPUTATIONAL BIOLOGY phagophore by conjugating with a phosphatidylethanolamine (PE) known functions of interacting with other proteins (13, 17). lipid catalyzed by the E3-like ATG5–ATG12–ATG16L1 complex Then, we purified a uniformly 15N-labeled STX17(142–228) during the action of autophagy (4, 5, 32–34). The PE-conjugated fragment that includes the entire Qa-SNARE motif (residues ATG8s are present both on the inner and outer membranes of the 167 to 224), and acquired its 1H-15N heteronuclear single- emerging closed autophagosome before fusion with the lysosome (4, quantum coherence (HSQC) spectrum (SI Appendix, Fig. S2A). 5). They are demonstrated to play crucial roles in autophagosome The small dispersion of the NMR peaks in the 1Hdimensionof biogenesis, autophagic cargo engulfment, autophagic vesicle trans- the 1H-15N HSQC spectrum together with the determined sec- port, and fusion of the autophagosome with the lysosome or endo- ondary structure of this STX17 Qa-SNARE region based on the 13 13 some by associating with relevant proteins that contain a short motif Cα and Cβ chemical shift values of each residue after backbone called the LC3-interacting region (LIR) in mammals (19, 31–33, chemical shift assignments indicated that the isolated STX17 Qa- 35–39). The canonical LIR motif contains a consensus core sequence SNARE motif is basically unstructured (SI Appendix,Fig.S2). ΘXXΓ (where Θ represents an aromatic Trp, Tyr, or Phe residue; Γ Interestingly, titration of the 15N-labeled STX17(142–228) with represents a bulky hydrophobic Leu, Ile, or Val residue; and X the unlabeled STX17(1–123) protein showed that a selected set of represents any amino acid residue) (31, 38, 40). Additional negatively peaks in the 1H-15N HSQC spectrum of STX17(142–228) under- charged serine/threonine phosphorylation sites and/or acidic residues went significant dose-dependent peak broadening or chemical preceding the LIR core sequence are also routinely found in typical shift changes (Fig. 1B), indicating that the STX17 Qa-SNARE LIR motifs, which are proven to regulate the interactions of LIR- region can specifically interact with the N-terminal Habc domain containing proteins with ATG8 family members (31, 38, 40, 41). In- of STX17. Further plotting of the peak broadening and amide triguingly, our previous study together with other groups’ reports backbone chemical shift changes as a function of residue number revealed that some LIR motifs, such as that of FYCO1, ankyrin-G, revealed that the significant perturbations are mainly rich in the and ankyrin-B, also include C-terminal extensions that can participate N-terminal part of the STX17 Qa-SNARE motif (residues 174 to in the interaction with ATG8 family orthologs following the core 194) (Fig. 1C), suggesting that this region is the major ΘXXΓ sequence (42–44). In particular, these C-terminal extensions for interacting with the STX17 Habc domain. not only can facilitate strong binding to ATG8s but also endow LIR- In contrast to that of STX17(142–228), the 1H-15N HSQC containing proteins with binding selectivity to different ATG8 spectrum of STX17(1–123) is well-dispersed, indicating that this orthologs (42–44). Notably, previous functional studies well- region constitutes an independently well-folded domain (Fig. 1D). demonstrated that the GABARAP subfamily is preferentially in- Further titration of 15N-labeled STX17(1–123) with unlabeled volved in the autophagosome–lysosome fusion process and the LC3s STX17(142–228) showed that the majority of peaks in the 1H-15N are unable to replace for the fusion between the HSQC spectrum underwent significant dose-dependent peak autophagosome and lysosome (19, 45), suggesting that some proteins broadening (Fig. 1D), confirming the existence of a direct inter- involved in the autophagosome–lysosome fusion process may also action between the Habc domain and Qa-SNARE motif of contain C-terminal extensions for selectively binding to the STX17. Strikingly, in the presence of STX17(142–228), a set of GABARAP subfamily. Interestingly, STX17 was recently reported to peaks appeared in the 1H-15N HSQC spectrum of STX17(1–123) contain two putative LIR motifs within its Qa-SNARE motif region, (Fig. 1D). Based on this observed NMR phenomenon and a series and can bind to ATG8 family proteins, especially LC3B and of NMR titration experiments using different truncation mutants GABARAP (30). However, how the two putative LIR motifs of of the STX17 Qa-SNARE motif, we further confirmed that the STX17 interact with ATG8 family proteins is still elusive. N-terminal region of the STX17 Qa-SNARE motif is responsible In this study, we biochemically and structurally character- for the interaction with STX17(1–123) (SI Appendix,Fig.S3). ized the key autophagosomal SNARE STX17 as well as its Unfortunately, due to the serious concentration-dependent peak

Li et al. PNAS Latest Articles | 3of12 Downloaded by guest on October 1, 2021 broadening that was likely induced by nonspecific self-associations the STX17 SNARE motif (Fig. 1 B–D). Importantly, additional (SI Appendix,Fig.S4A), we were unable to achieve the backbone NMR characterizations uncovered that in contrast to SNAP29 and assignments for STX17(1–123). However, in the presence of a VAMP8 (SI Appendix,Fig.S6), GABARAP can compete against saturated amount of STX17(142–228) protein, we were able to the N-terminal Habc domain for binding to the SNARE motif of finish the backbone assignments for the remaining NMR peaks (SI STX17, thereby easily relieving the autoinhibited state of STX17 Appendix,Fig.S4B). Interestingly, we found that the newly (Fig. 2E). appeared NMR peaks arose from residues 94 to 121, which are Then, we sought to understand how the STX17 SNARE motif located in the predicted extreme C-terminal α-helix of the Habc recognizes the mammalian ATG8 orthologs. We chose domain (SI Appendix,Fig.S1A), but were demonstrated to be GABARAP and LC3A as two representatives of the GABARAP unstructured in the presence of STX17(142–228) based on our subfamily and the LC3 subfamily, respectively, and carefully NMR analysis (SI Appendix,Fig.S4C). Taken together, all these characterized their interactions with STX17(142–228). NMR- data clearly demonstrated that the Qa-SNARE motif of STX17 based analyses showed that both GABARAP and LC3A can can directly bind to the STX17 Habc domain and alter its con- interact with STX17(142–228) (Fig. 3A and SI Appendix, Fig. formation by partially unfolding its extreme C-terminal α-helix. S13A), and the major binding sites on STX17 are located within Thus, the cytoplasmic region of STX17 may adopt an autoinhibited the N-terminal part (residues 170 to 191) of the STX17 Qa- closed conformation imposed by an intramolecular interaction be- SNARE motif (Fig. 3B and SI Appendix, Fig. S13B). Notably, tween its Habc domain and the Qa-SNARE motif. Notably, the the binding sites of the STX17 SNARE motif for interacting with STX17 Qa-SNARE motif only showed a negligible weak interaction mammalian ATG8 orthologs and the STX17 Habc domain are with the full-length SNAP29 or the VAMP8 R-SNARE motif highly overlapped (Figs. 1 B and C and 3 A and B and SI Ap- (residues 8 to 66) proteins based on our NMR titration results (SI pendix, Fig. S13 A and B), therefore mechanistically explaining Appendix,Fig.S5). Importantly, further NMR analyses revealed why mammalian ATG8 orthologs can compete with the that neither the full-length SNAP29 nor the VAMP8 R-SNARE N-terminal Habc domain for binding to the SNARE motif of motif alone was able to release the autoinhibited state of STX17 (SI STX17. Interestingly, detailed sequence analysis showed that the Appendix,Fig.S6). Consistent with these NMR-based analyses (SI STX17 SNARE region contains two putative LIR motifs, Appendix,Figs.S5andS6), further analytical gel filtration “WETL” (residues 172 to 175) and “FSLL” (residues 189 to chromatography-based assays revealed that full-length SNAP29 192), although the critical aromatic Phe residue in the second cannot obviously interact with VAMP8(1–75) that includes the putative LIR motif is not strictly conserved in mammals (SI entire cytoplasmic region of VAMP8 nor with STX17(1–228) (SI Appendix, Fig. S1A). Further NMR-based analyses revealed that Appendix,Fig.S7A and B), and there is an extremely weak inter- the NMR resonances of the first putative LIR motif show much action between STX17(1–228) and VAMP8(1–75) (SI Appendix, more significant changes than those of the second putative LIR Fig. S7C). As expected, when mixing these three proteins together, region when titrated with GABARAP or LC3A (Fig. 3 A and B we can readily detect a ternary SNARE complex containing and SI Appendix,Fig.S13A and B), suggesting that only the first SNAP29 full-length, VAMP8(1–75), and STX17(1–228) (SI Ap- putative LIR motif may directly participate in the interaction with pendix,Fig.S7D and E). Therefore, unlike the neuronal SNARE GABARAP or LC3A. To further test whether these two putative complex, the formation of a binary STX17–SNAP29 (Qa/Qbc) LIR motifs are directly involved in the GABARAP or LC3A bind- t-SNARE complex during the autophagic SNARE complex as- ing, we mutated the two crucial aromatic residues (W172 and F189) sembly process is unfeasible in vitro. within the two putative LIR motifs of STX17(142–228) and con- structed three mutants, STX17(142–228) W172Q, STX17(142–228) The STX17 SNARE Region Contains One LIR Motif That Can Selectively F189Q, and the STX17(142–228) W172Q/F189Q double mutant. Bind to Mammalian ATG8 Orthologs. Since mammalian ATG8 Then, we used these three mutants together with the wild-type family proteins, especially LC3B and GABARAP, were reported STX17(142–228) and quantitatively compared their interactions to interact with the STX17 Qa-SNARE region (30), we wondered with GABARAP and LC3A using ITC-based assays (Fig. 3 C–F and whether these mammalian ATG8 orthologs might regulate the SI Appendix,Fig.S13C–F). The obtained ITC results showed that closed conformation of STX17. To test this hypothesis, we purified the W172Q mutation dramatically reduces and totally abolishes the two STX17 fragments, STX17(142–228) and STX17(1–228), that interaction of STX17(142–228) with GABARAP and LC3A, re- include the entire cytoplasmic region of STX17 and should adopt spectively (Fig. 3 C and D and SI Appendix,Fig.S13C and D), while an autoinhibited state, and investigated their interactions with the the F189Q mutation does not affect the binding of STX17(142–228) six mammalian ATG8 homologs. Using analytical gel filtration to GABARAP and LC3A (Fig. 3 C and E and SI Appendix,Fig. chromatography-based analyses, we found both STX17(142–228) S13 C and E), confirming that only the first putative LIR motif of and STX17(1–228) can directly interact with all of the six mam- STX17 is directly involved in the interactions with GABARAP and malian ATG8 orthologs (Fig. 2 A and B and SI Appendix,Figs.S8 LC3A (Fig. 3 A and B and SI Appendix,Fig.S13A and B). Sur- and S9). Further quantitative analyses of the interactions of these prisingly, the STX17(142–228) W172Q and W172Q/F189Q mutants two STX17 fragments with different ATG8 homologs using iso- still displayed some residual binding abilities to GABARAP but not thermal titration calorimetry (ITC) measurements revealed that LC3A (Fig. 3 D and F and SI Appendix,Fig.S13D and F), and they STX17 can selectively bind to six mammalian ATG8 orthologs can weakly bind to GABARAP with similar Kd values, ∼19 and ∼21 with distinct binding affinities (Fig. 2 C and D and SI Appendix, μM, respectively (Fig. 3 D and F), implying that, except for the ca- Figs. S10–S12). In particular, STX17 preferentially binds to nonical hydrophobic LIR core sequence, there are additional struc- GABARAP subfamily members (GABARAP, GABARAPL1, tural features that may contribute to the interaction of STX17 with and GABARAPL2) rather than LC3 subfamily members (LC3A, GABARAP but not LC3A. Finally, using analytical ultracentrifuga- LC3B, and LC3C) (Fig. 2 C and D and SI Appendix,Figs.S10and tion analyses, we further elucidated that STX17(142–228) and S11). Notably, the STX17(1–228) fragment displays a relatively GABARAP both form monomers in solution and, importantly, they weaker binding affinity toward ATG8 family members than that of caninteractwitheachothertoform a 1:1 stoichiometric complex STX17(142–228) based on our ITC analyses (Fig. 2 C and D and (Fig. 3G), consistent with our notion that the Qa-SNARE region of SI Appendix, Figs. S10 and S11), suggesting that the STX17 only contains one LIR motif. STX17 N-terminal Habc region somehow interferes with the in- teractions between the STX17 SNARE region and ATG8 ortho- The Overall Structure of the STX17 LIR Motif in Complex with logs, consistent with our aforementioned observation that the GABARAP. To further elucidate the molecular mechanism gov- N-terminal Habc domain of STX17 can directly interact with erning the interaction between the STX17 LIR motif and

4of12 | www.pnas.org/cgi/doi/10.1073/pnas.2006997117 Li et al. Downloaded by guest on October 1, 2021 Fig. 2. GABARAP can directly bind to STX17 and relieve the autoinhibited conformation of STX17. (A and B) Analytical gel filtration chromatography analyses 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 of GABARAP 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 with STX17(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 the

ITC machine. BIOPHYSICS AND COMPUTATIONAL BIOLOGY

mammalian ATG8 proteins, we sought to determine their com- that the overall interaction modes of the extended LIR motifs from plex structures. Initially, we purified the STX17(142–228)– STX17, FYCO1, and AnkB/G toward ATG8 family proteins are GABARAP complex to conduct a crystal screening but, unfor- very similar but, strikingly, only the STX17 LIR includes a tunately, we only obtained crystals with poor diffractions. Given C-terminal extension with a 310 helix (SI Appendix,Fig.S14B). that the STX17(142–228) fragment is basically unstructured, we further narrowed down the N- and C-terminal boundaries of The Molecular Interface of the STX17 LIR–GABARAP Complex. De- STX17 and chose a STX17(167–188) fragment, which includes tailed structural analysis of the binding interface of the STX17 the entire proven LIR motif but lacks the second putative LIR LIR–GABARAP complex revealed that the binding between sequences (SI Appendix, Fig. S1A). Fortunately, using the puri- STX17 LIR and GABARAP is mediated by extensive hydro- fied STX17(167–188)–GABARAP complex, we obtained good phobic contacts and polar interactions (Fig. 4 B and C and SI crystals that diffracted to 2.0-Å resolution. The crystal structure of Appendix, Fig. S14C). In particular, the aromatic side chain the STX17 LIR–GABARAP complex was determined using the of W172 of STX17 LIR occupies a hydrophobic pocket of molecular replacement method (SI Appendix,TableS1). In the final GABARAP mainly assembled by the hydrophobic side chains of refined structural model, each asymmetric unit contains four I21, P30, L50, and F104 as well as the aliphatic side chain of K48 STX17 LIR–GABARAP complex molecules, and each STX17 and, meanwhile, the side-chain group of STX17 W172 also forms LIR–GABARAP complex has a 1:1 stoichiometry (Fig. 4A), in line a hydrogen bond with the side chain of E17 located at the α2- with our biochemical result (Fig. 3G). As expected, the GABARAP helix of GABARAP (Fig. 4 B and C). In parallel, the hydro- molecule in the complex structure adopts a typical ATG8 homolog phobic side chains of L175, L179, and L182 from STX17 LIR protein architecture consisting of two N-terminal α-helices followed pack against a hydrophobic patch that is situated at the β2/α3- by a ubiquitin-like core that is assembled by a four-stranded β-sheet groove and formed by the side chains of the Y49, V51, P52, L55, together with two α-helices (Fig. 4A). In the complex structure, the F60, F62, and L63 residues of GABARAP (Fig. 4 B and C). GABARAP-bound STX17 LIR motif is mainly composed of two Moreover, the backbone oxygen of STX17 E170 forms a strong parts: an N-terminal extended structure formed by the canonical hydrogen bond with the side chain of the K48 residue located at LIR core containing the signature ΦXXΨ (WETL) sequences and the β2-strand of GABARAP, and the backbone oxygen and an additional C-terminal 310-helix extension (Fig. 4 A and B and SI amide group of STX17 E173 form two backbone hydrogen bonds Appendix,Fig.S1A), in agreement with our aforementioned ITC with the K48 and L50 residues of GABARAP (Fig. 4C). In ad- results (Fig. 3 D and F). The entire STX17 LIR motif packs ex- dition, the STX17 LIR–GABARAP complex is further stabilized tensively with a solvent-exposed elongated groove mainly formed by by two charge–charge interaction networks, one of which is lo- the α1-, α2-, and α3-helices together with the β2-strand of cated at the N-terminal region of GABARAP and is assembled GABARAP, burying a total surface area of ∼778 Å2 (Fig. 4 A and by the negatively charged STX17 E170 residue coupled with the B and SI Appendix,Fig.S14A). Intriguingly, structural comparisons positively charged H9 and K47 residues of GABARAP, while the of the STX17 LIR–GABARAP complex with currently known other is formed between the negatively charged E173, D178, and complexes of ATG8s bound with unconventional LIR motifs that E181 residues of STX17 LIR and positively charged K66 and contain C-terminal extensions, such as the FYCO1 LIR–LC3A R67 residues of GABARAP (Fig. 4 B and C and SI Appendix, complex ( [PDB] ID code 5CX3) (42), AnkB Fig. S14C). Notably, all these key interface residues of STX17 LIR–GABARAP complex (PDB ID code 5YIR), and AnkG and GABARAP are highly conserved during evolution (SI Ap- LIR–GABARAPL1 complex (PDB ID code 5YIP) (43), revealed pendix, Fig. S1). Using ITC and coimmunoprecipitation (co-IP)

Li et al. PNAS Latest Articles | 5of12 Downloaded by guest on October 1, 2021 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 HSQC spectra 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 differences and 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 ratio of 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 chemical shift 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 STX17 are 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 of STX17(142–228) (black), GABARAP (red), and STX17(142–228)–GABARAP complex (blue). These results demonstrate that STX17(142–228) and GABARAP form monomers and interact with each other to form a 1:1 stoichiometric complex.

analyses, we further verified the interactions observed in the based assays demonstrated that the GABARAP K47T/L55V/ structure of the STX17 LIR–GABARAP complex. Consistent F62K triple mutant has a comparable binding affinity for with our structural data, the ITC results showed that individual STX17(142–228) to that of LC3A and, conversely, the V58L/ point mutations of the key interface residues either from STX17 K65F double mutant of LC3A has a much increased binding or GABARAP, such as the E170A, W172Q, D178R, and L179Q ability for STX17(142–228) (SI Appendix, Fig. S17). In all, the mutations of STX17(142–228) or the K47E, K48E, L55A, L63Q, identification of these nonconserved interface residues among K66E, and R67E mutations of GABARAP, all largely reduce or different mammalian ATG8 orthologs not only provided a completely disrupt the interaction between STX17(142–228) and mechanistic explanation for the selective binding of STX17 LIR GABARAP (Fig. 4D and SI Appendix, Fig. S15). Importantly, toward different mammalian ATG8 orthologs but also rationalized consistent with our in vitro ITC results, further co-IP experi- our aforementioned biochemical results that the STX17(142–228) ments revealed that point mutations of key interface residues W172Q and W172Q/F189Q mutants can weakly bind to including the K48E and R67E mutations of GABARAP and the GABARAP but not to LC3A that lacks the crucial bulky hydro- W172Q and D178R mutations of STX17 all significantly atten- phobic L55 and F62 residues for interacting with the C-terminal 310- uate or essentially abolish the interaction between full-length helix extension of STX17 (Fig. 3 D and F and SI Appendix,Fig. STX17 and GABARAP in cotransfected cells (Fig. 4E). Nota- S13 D and F). bly, further detailed structure-based sequence alignment and structural comparison analyses showed that several key binding- The Biochemical and Structural Properties of the STX17–SNAP29–VAMP8 interface residues are quite different among the six mammalian SNARE Complex. Given that GABARAP can occupy the N-terminal ATG8 orthologs (SI Appendix, Fig. S16). For instance, the resi- part of the STX17 SNARE motif and form a stable complex with dues corresponding to the bulky hydrophobic L55 and F62 in STX17, next we wanted to know whether GABARAP may affect GABARAP, which are critical for the hydrophobic interaction the assembly of the STX17–SNAP29–VAMP8 SNARE complex. with the C-terminal 310 helix (SI Appendix, Fig. S16B), are a Using analytical gel filtration chromatography-based analyses, we relatively smaller Val residue in LC3A and LC3B and a polar Lys showed that the Qb- and Qc-SNARE motifs of SNAP29 alone are or Ser residue in LC3s, respectively (SI Appendix, Fig. S16A); the unable to interact with or disturb the GABARAP–STX17 complex residue corresponding to the positively charged K47 in (SI Appendix,Fig.S18A); however, in the presence of an additional GABARAP is a neutral Thr residue in LC3s, and the residue VAMP8 R-SNARE motif, a stable SNARE complex containing the corresponding to the positively charged R66 in GABARAP is a STX17 Qa-SNARE motif, the VAMP8 R-SNARE motif, as well as neutral Ser residue in LC3C (SI Appendix, Fig. S16A). Consistent the Qb- and Qc-SNARE motifs of SNAP29, was readily formed (SI with these sequence- and structure-based analyses, further ITC- Appendix,Fig.S18B and C), indicating that SNAP29 and VAMP8

6of12 | www.pnas.org/cgi/doi/10.1073/pnas.2006997117 Li et al. Downloaded by guest on October 1, 2021 BIOPHYSICS AND COMPUTATIONAL BIOLOGY

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

together can easily compete with GABARAP for binding to STX17 indicated by its CD spectrum (Fig. 5A). It is noteworthy that the to assemble the STX17–SNAP29–VAMP8 SNARE complex. Since isolated SNARE region of STX17 is intrinsically disordered based the membrane fusion between the double-membraned autophago- on the CD analysis (Fig. 5A), consistent with the aforementioned some and single-membraned lysosome is morphologically distinct NMR results (SI Appendix,Fig.S2). Interestingly, further CD-based from the conventional fusion event between two single-membraned analysis revealed that with increasing temperature, this autophagic vesicles, we wondered whether this autophagic STX17–SNAP29– SNARE complex undergoes unfolding with a melting temperature VAMP8 SNARE complex has striking biochemical properties. To (Tm)of∼85 °C (Fig. 5B), which is a little higher than that of the late test this hypothesis, we first purified this autophagic SNARE endosomal SNARE complex (Tm 78 °C) but slightly lower than its complex, which includes the SNARE motifs of STX17 and VAMP8 early endosomal (Tm 87 °C) and neuronal (Tm 90 °C) counterparts together with their short neck regions, and the Qb- and Qc-SNARE (46–48). Subsequent reduction of the temperature of the sample led motifs of SNAP29, and then used circular dichroism (CD) spec- to the initiation of refolding at a much lower temperature of ∼51 °C troscopy to examine its secondary structure features and thermal and further cooling to 10 °C resulted in a partial refolding of the stability. In contrast to that of the isolated SNARE regions of original α-helical content (Fig. 5B), showing the characteristic STX17 and VAMP8 as well as the full-length SNAP29, the SNARE unfolding–refolding hysteresis of a typical SNARE core complex complex showed significant characteristic α-helical content, as (28, 49).

Li et al. PNAS Latest Articles | 7of12 Downloaded by guest on October 1, 2021 We also solved the crystal structure of this autophagic SNARE bundle (Fig. 5C and SI Appendix, Fig. S19). Notably, a similar core complex (SI Appendix, Table S1). As expected, this auto- overall architecture was also observed in a previously reported phagic SNARE complex forms a four-helix bundle with all four structure of the STX17–SNAP29–VAMP8 SNARE complex helices aligned in parallel, and STX17 and VAMP8 each con- with slightly different protein boundaries (16). Interestingly, the tribute one helix while SNAP29 contributes two helices to the electrostatic 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 SNARE complex. (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 complex formed by 16 layers of interacting amino acid side chains.

8of12 | www.pnas.org/cgi/doi/10.1073/pnas.2006997117 Li et al. Downloaded by guest on October 1, 2021 showed two highly negatively charged patches (Fig. 5D), which lysosome fusion process and what the potential in vivo function is are on opposite sides of the surface and are implicated in the of the conformational change of STX17 Habc induced by the binding to the NSF-mediated SNARE-disassembly machinery Qa-SNARE motif remain to be elucidated. (28, 50). Similar to other known SNARE complexes, the interior Our study showed that SNAP29 alone is unable to open the of this autophagic SNARE complex is formed by 16 layers of closed conformation of STX17 to form a stable STX17–SNAP29 interacting amino acid side chains that are mostly hydrophobic, (Qa/Qbc) t-SNARE complex in vitro (SI Appendix, Figs. S6A and the hydrophilic 0 layer is formed by three Gln residues (Q196 and S7B). However, mammalian ATG8 orthologs, such as of STX17 and Q84 and Q230 of SNAP29) and one Arg resi- GABARAP, could easily release the autoinhibited conformation due (R37 of VAMP8) (Fig. 5E and SI Appendix,Fig.S19). De- of STX17 by competitively binding to the N-terminal region of tailed structural analyses of the other 15 layers revealed that the STX17 SNARE motif and form a stable binary complex with the −fifth, −first, first, fourth, and eighth layers are all formed by STX17. Since previous relevant functional studies have well- four hydrophobic side chains, whereas the −seventh, −sixth, −fourth, demonstrated that the inactivation of all six mammalian ATG8 second, third, fifth, and seventh layers are all composed of three orthologs or the mammalian ATG8 conjugation machinery sig- hydrophobic side chains and one polar side chain of a Ser or Thr nificantly attenuates the recruitment of STX17 to the autopha- residue, of which the hydroxyl group throughout points to the out- gosome as well as the fusion between the autophagosome and side of the layer (Fig. 5E). Notably, the arrangements of the side lysosome (19, 20), our work may provide direct structural evi- chains in the −third, −second, and sixth layers are highly asymmetric dence for the essential function of mammalian ATG8s in me- (Fig. 5E). Next, we sought to use the glutathione S- diating the autophagosome–lysosome fusion process. However, (GST)–fusion protein pull-down assay to verify the contributions given that STX17 is exclusively recruited to the external mem- of different intact layers to the assembly of the autophagic SNARE brane of the autophagosome instead of the phagophore that is complex. We individually disrupted the intact −seventh, −fifth, 0, also decorated with mammalian ATG8s (13, 20), the sole in- fourth, and seventh layers of the SNARE complex by mutation of teractions between STX17 and mammalian ATG8s are likely key residues involved in the layer formation either from STX17 or insufficient to accomplish the recruitment of STX17 to the SNAP29. The W172Q (−seventh layer), L179Q (−fifth layer), and emerging autophagosome rather than the phagophore. Inter- Q196L (0 layer) mutations of STX17 as well as the L213Q (−fifth estingly, a recent study showed that the autophagy-related small layer) and Q230L (0 layer) mutations of SNAP29 essentially abol- GTPase IRGM in its active form can directly associate with ished the SNARE core complex formation, whereas the A210Q STX17 by binding to its two transmembrane domains (30), which (fourth layer) and L221Q (seventh layer) mutations of STX17 only was proven to be essential for the translocation of STX17 to E SI partially weakened the core complex formation (Fig. 5 and autophagic membranes (13). Unfortunately, we were unable to BIOPHYSICS AND COMPUTATIONAL BIOLOGY Appendix,Fig.S20), suggesting that the N-terminal and central layers obtain soluble IRGM proteins either from Escherichia coli or are much more important than the C-terminal layers during the insect cells, thereby preventing detailed biochemical and struc- assembly of the autophagic SNARE complex, in keeping with the tural characterizations. Additional work is required to elucidate zipper-like mode for canonical SNARE complex assembly (28, 51). the detailed mechanism governing the temporal and spatial In addition, the four-helix bundle structure of this autophagic regulation of the interaction of STX17 with mammalian ATG8s SNARE complex is further stabilized by extensive surface interac- as well as the recruitment of STX17 to the autophagosome. tions between different helices (SI Appendix, Figs. S17 and S21). Furthermore, our systemic biochemical and structural char- acterizations revealed that the key binding-interface residues of Discussion STX17 involved in GABARAP binding and the STX17– In this study, we uncovered that the STX17 Qa-SNARE motif SNAP29–VAMP8 SNARE complex assembly are heavily over- can directly interact with its N-terminal Habc domain, suggesting lapped (Figs. 4C and 5E and SI Appendix, Figs. S1A and S19B), that, in isolation, STX17 adopts a closed conformation. Con- such as the W172 and L175 residues of STX17. Therefore, due to sidering the poor quality of the 1H-15N HSQC spectrum of the the potential steric exclusion, once the STX17–SNAP29– STX17 Habc region in the presence of the STX17 SNARE motif VAMP8 SNARE complex is formed, GABARAP and likely (Fig. 1D and SI Appendix, Fig. S3), we sought to use X-ray other ATG8 orthologs are unable to associate with this auto- crystallography to solve the autoinhibited structure of STX17 phagic SNARE complex (SI Appendix, Fig. S18 B and C). In- Habc in complex with the Qa-SNARE motif. Unfortunately, terestingly, based on careful structural analyses (Fig. 4C and SI after numerous trials, we failed to obtain good crystals for Appendix, Fig. S22A), we rationally designed and obtained a structure determination, presumably due to the dynamic nature STX17 D178R point mutation, which disrupted the interaction of this unstable complex, as indicated by our NMR analyses of STX17 with GABARAP without affecting the assembly and (Fig. 1 B and D). Hence, further studies are required to elucidate the stability of the STX17–SNAP29–VAMP8 SNARE complex the detailed molecular mechanism underlying the interaction (SI Appendix, Figs. S15B and S22B). Therefore, this mutation between the Habc domain and the Qa-SNARE motif of STX17. may potentially be useful for the future functional study of Strikingly, a similar intramolecular interaction between the STX17 as well as the dissection of different roles mediated by the N-terminal Habc domain and the C-terminal Qa-SNARE motif STX17–ATG8 ortholog interaction and this STX17-containing was observed in Syntaxin1, a neuronal Qa-SNARE protein in- SNARE complex for autophagosome–lysosome fusion. volved in synaptic membrane fusion (52). However, unlike that In summary, we proposed a model depicting three different of Syntaxin1, the binding of the STX17 SNARE motif to its Habc states of STX17 in cooperation with mammalian ATG8s as well domain can induce a partial unfolding of the extreme C-terminal as the autophagy-related SNARE proteins SNAP29 and VAMP8 α-helix of the STX17 Habc domain (Fig. 1D and SI Appendix, during the autophagosome–lysosome fusion process (Fig. 6). In Fig. S4C). Importantly, previous studies of Syntaxin1 had well- this model, STX17 alone is in an autoinhibited closed state and, demonstrated that the closed conformation of Syntaxin1 is es- particularly, its Habc region somehow packs with the N-terminal sential for its interaction with Munc18-1, a major regulator of part of its Qa-SNARE motif to stabilize the intrinsically disor- fusion, and represents a critical intermediate for dered SNARE motif of STX17, thereby preventing its unnecessary neuronal exocytosis (52). Similarly, STX17 was reported to in- degradation or interactions with other proteins on the autopha- teract with the HOPS tethering complex through the Munc18- gosome before assembling the STX17–SNAP29–VAMP8 SNARE like subunit Vps33 for mediating autophagosome–lysosome fu- complex (Fig. 6). However, in the presence of GABARAP/LC3 sion (15, 17, 53). However, whether HOPS binds to a closed or family proteins, the mammalian ATG8 ortholog can competitively an open conformation of STX17 during the autophagosome– bind to the N-terminal part of the STX17 Qa-SNARE motif that

Li et al. PNAS Latest Articles | 9of12 Downloaded by guest on October 1, 2021 contains an extended LIR motif and abolish the autoinhibited SNAP29–VAMP8 SNARE complex was further purified using a Superdex conformation of STX17 (Fig. 6), thereby releasing the N-terminal 75 size-exclusion column equilibrated with a column buffer containing 20 mM Habc domain of STX17 and inducing its conformational rear- Tris·HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, and 1 mM ethylenediaminetetraacetic rangement. Then, relevant tethering factors, such as the HOPS acid (EDTA). Uniformly 15N- or 15N/13C-labeled proteins were prepared by growing complex, ATG14, and EPG5, are recruited, which in turn work 15 bacteria in M9 minimal medium using NH4Cl (Cambridge Isotope Labora- with STX17 and promote the further recruitment of cytosolic Qbc- 15 13 tories; NLM-467) as the sole nitrogen source or NH4Cl and [ C6]glucose SNARE SNAP29 and bring the VAMP8-residing lysosome into (Cambridge Isotope Laboratories; CLM-1396) as the sole nitrogen and carbon close proximity to the autophagosome (Fig. 6). Finally, mediated sources, respectively. by their respective SNARE motifs, STX17, SNAP29, and VAMP8 assemble into the autophagic SNARE complex to clamp the NMR Spectroscopy. The stable isotope-labeled protein samples for NMR membranes together and initiate the fusion between the auto- studies were concentrated to ∼0.1 mM for titration experiments and phagosome and lysosome, eventually leading to the formation of ∼0.6 mM for backbone resonance assignment experiments in 50 mM po- the autolysosome and the subsequent degradation of the enclosed tassium phosphate buffer containing 50 mM NaCl (pH 6.5) and 1 mM DTT. materials (Fig. 6). NMR spectra were acquired at 25 °C on an Agilent 800-MHz spectrometer equipped with an actively z gradient-shielded triple-resonance cryogenic Materials and Methods probe. Backbone resonance assignments of STX17(142–228) and the STX17 SNARE-bound Habc domain were achieved using a suite of hetero- Protein Expression and Purification. Different DNA fragments encoding hu- nuclear correlation experiments including HNCO, HNCA, CA(CO)NH, man STX17, SNAP29, VAMP8, and ATG8s and other related DNA fragments HNCACB, and CBCA(CO)NH using a 15N/13C-labeled protein sample together were amplified by PCR from the full-length human complementary DNA with a three-dimensional 15N-separated NOESY (54). (cDNA). All these fragments were cloned into in-house modified versions of the pET32a vector for recombinant protein expression. For the coimmuno- precipitation assay, full-length STX17 and GABARAP DNA fragments were Analytical Gel Filtration Chromatography. Analytical gel filtration chroma- cloned into pmCherry-C1 and pEGFP-C1 vectors, respectively. All of the point tography was carried out on an AKTA FPLC System (GE Healthcare). Protein mutations of STX17, SNAP29, GABARAP, and LC3A used in this study were samples were loaded onto a Superose 12 10/300 GL column (GE Healthcare) · created using the standard PCR-based mutagenesis method, further checked equilibrated with a buffer containing 20 mM Tris HCl (pH 7.5), 100 mM NaCl, by PCR screen using 2× Taq Master Mix (Vazyme Biotech) and and 1 mM DTT. confirmed by DNA sequencing. Recombinant proteins were expressed in BL21 (DE3) E. coli cells induced by Isothermal Titration Calorimetry Assay. ITC measurements were carried out on 100 μM isopropyl β-D-1-thiogalactopyranoside at 16 °C. The bacterial cell a MicroCal PEAQ-ITC calorimeter or an automated system (Malvern) at 25 °C. pellets were resuspended in binding buffer (50 mM Tris, pH 7.9, 500 mM For each ITC experiment in this study, the protein samples in the cell and in NaCl, 5 mM imidazole), and then lysed by the FB-110XNANO homogenizer the syringe were exchanged into the same buffer condition containing machine (Shanghai Litu Machinery Equipment Engineering). Then the lysate 20 mM Tris (pH 7.5), 100 mM NaCl, and 1 mM DTT using a HiPrep 26/10 μ was centrifuged at 35,000 × g for 30 min to remove debris. His -tagged desalting column. The titration processes were performed by injecting 40- L 6 ∼ μ ∼ μ proteins were purified using Ni2+-NTA agarose (GE Healthcare) affinity aliquots of the syringe sample ( 500 M) into the cell sample ( 50 M) at chromatography followed by size-exclusion chromatography (Superdex 75 time intervals of 2 min to ensure that the titration peak returned to base- or 200 column; GE Healthcare) in a buffer containing 20 mM Tris·HCl (pH line. In addition, relevant reference control experiments, in which the pro- 7.5), 100 mM NaCl, and 1 mM dithiothreitol (DTT). Purities and molecular tein samples in the syringe are titrated into the control buffers, were also masses were verified by sodium dodecyl sulfate/polyacrylamide gel electro- conducted. For each ITC dataset, the reference data would be subtracted phoresis (SDS/PAGE) analysis. from the raw data to obtain the net result for the final fitting analysis. The To obtain the STX17 LIR–GABARAP complex proteins used for crystalli- titration data were analyzed using the Malvern MicroCal PEAQ-ITC analysis zation, GST-STX17(167–188) and His-GABARAP were first copurified by glu- program. The Kd error is the fitted error obtained from the data analysis tathione Sepharose 4B (GE Healthcare) affinity chromatography. Then, the software when using the one-site binding model to fit the ITC data. N-terminal tags were cleaved by 3C protease, and the GST tag was further removed by glutathione Sepharose 4B affinity chromatography. Finally, the Analytical Ultracentrifugation. Sedimentation velocity experiments were STX17(167–188)–GABARAP complex was further purified on a Superdex performed on a Beckman XL-I analytical ultracentrifuge equipped with an 75 size-exclusion column that was equilibrated with a column buffer con- eight-cell rotor at 42,000 rpm at 20 °C. The partial specific volume of dif- taining 20 mM Tris·HCl (pH 7.5), 100 mM NaCl, and 1 mM DTT. For the ferent protein samples and the buffer density were calculated using the STX17–SNAP29–VAMP8 SNARE core complex proteins used for crystalliza- program SEDNTERP (http://www.rasmb.org/). The final sedimentation ve- tion, Trx-STX17(142–228), MBP-SNAP29(40–126), GB1-SNAP29(191–258), and locity data were analyzed and fitted to a continuous sedimentation coeffi- Trx-VAMP8(8–75) were first copurified by Ni2+-NTA agarose (GE Healthcare) cient distribution model using the program SEDFIT (55). The fitting results affinity chromatography followed by size-exclusion chromatography were further output to Origin 9.0 software and aligned with each other. (Superdex 200 column). Fractions containing the SNARE complex were col- lected and digested by 3C protease, loaded onto a MonoQ 10/10 ion- Protein Crystallization and Structural Elucidation. Crystals of the STX17 LIR– exchange column (GE Healthcare), and eluted with a linear NaCl gradient GABARAP complex and autophagic STX17–SNAP29–VAMP8 SNARE complex up to 500 mM to remove N-terminal tags. Finally, the autophagic STX17– were 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 mammalian ATG8s, autophagic SNARE proteins SNAP29 and VAMP8, and relevant tethering factors for facilitating the membrane fusion process between the auto- phagosome and lysosome in macroautophagy.

10 of 12 | www.pnas.org/cgi/doi/10.1073/pnas.2006997117 Li et al. Downloaded by guest on October 1, 2021 Specifically, crystals of the STX17 LIR–GABARAP complex were formed about path length of 0.5 mm was used. CD spectra were obtained by measuring the 1 wk after the freshly purified STX17 LIR–GABARAP complex (10 mg/mL in purified wild type or mutants of the autophagic STX17–SNAP29–VAMP8 20 mM Tris·HCl, pH 7.5, 100 mM NaCl, 1 mM DTT) was mixed with an equal SNARE core complex at a concentration of 20 μM in a buffer containing volume of reservoir solution containing 0.12 M alcohols, 50% (vol/vol) pre- 40 mM sodium phosphate buffer (pH 6.5). For the thermal melting experi- cipitant Mix 4, and buffer system 3 at pH 8.5 from the Morpheus Screen Kit ment, the wavelength was set to 222 nm and the temperature from 10 to – – (Molecular Dimensions). Meanwhile, crystals of the STX17 SNAP29 VAMP8 105 °C. Then the unfolding measurement was started by gradually increas- · SNARE complex (20 mg/mL in 20 mM Tris HCl, pH 7.5, 100 mM NaCl, 1 mM ing the temperature to 105 °C at a rate of 30 °C/h. Subsequently, the tem- DTT, 1 mM EDTA) were grown from buffer containing 2.5 M ammonium perature for refolding was again lowered to 10 °C at a rate of 30 °C/h. nitrate (pH 4.6) and 0.1 M sodium acetate trihydrate. Before the diffraction experiments, glycerol as appropriate was added as the cryoprotectant. X-ray GST Pull-Down Assay. Direct interactions between different Syntaxin17, datasets were collected at beamline BL17U1 or BL19U1 of the Shanghai SNAP29, and VAMP8 proteins were assayed in PBS (pH 7.4). The Syntaxin17 Synchrotron Radiation Facility (SSRF) (56). The diffraction data were pro- cessed and scaled using HKL2000 (57). fragment (residues 142 to 225/228) was tagged with GST, while the SNAP29 The phase problem of the STX17 LIR–GABARAP complex was solved by the fragment (residues 40 to 126), SNAP29 fragment (residues 191 to 258), and molecular replacement method using the modified structure of GABARAP VAMP8 fragment (residues 8 to 66/71) were tagged with MBP-His6, GB1-His6, (PDB ID code 5YIR) with PHASER (58). Meanwhile, the phase problems of the and Trx-His6, respectively. Fifty micrograms of GST-tagged proteins and His- STX17–SNAP29–VAMP8 SNARE complex were solved by the molecular re- tagged proteins was mixed at a molar ratio of 1:2 in 1 mL of the assay buffer. placement method using the crystal structure of the endosomal SNARE core The GST-STX17–SNAP29–VAMP8 complexes were pelleted by adding 30 μL complex (PDB ID code 1GL2) as the search model. All initial structural models fresh glutathione Sepharose 4B (GE Healthcare) beads. The pellets were were rebuilt manually using Coot (59) and then refined using REFMAC (60) washed six times with 1 mL of the assay buffer, and subsequently eluted or PHENIX (61). The qualities of the final model were validated by MolPro- with 60 μL of 30 mM glutathione buffer. Ten microliters of each eluted bity (62). The final refinement statistics of the solved structures in this study sample was separated by 12% SDS/PAGE and analyzed using Coomassie blue are listed in SI Appendix, Table S1. All of the structure figures were prepared staining or Western blot. using the program PyMOL (https://pymol.org/2/). Data Availability. The coordinates and structure factors of the STX17 LIR– Coimmunoprecipitation Assay. HEK293T cells transiently expressing proteins GABARAP complex and the STX17–SNAP29–VAMP8 SNARE complex repor- were harvested, washed with phosphate-buffered saline (PBS) buffer, and ted in this paper have been deposited in the Protein Data Bank (PDB ID lysed for 1 h at 4 °C in lysis buffer containing 50 mM Tris·HCl (pH 7.8), 50 mM codes 7BV4 and 7BV6, respectively). NaCl, 0.4% Nonidet P-40, 0.5 mM phenylmethanesulfonyl fluoride, and protease inhibitor mixture (AMRESCO). Lysates were centrifuged and then ACKNOWLEDGMENTS. We thank SSRF BL17U1 and BL19U1 for X-ray supernatants were incubated with the appropriate antibody pretreated with beamtime, Dr. Jianchao Li for help with X-ray diffraction data collection,

rProtein G agarose (Invitrogen) for 3 h with rotation at 4 °C. Precipitated BIOPHYSICS AND COMPUTATIONAL BIOLOGY and Prof. Jiahuai Han for the full-length Syntaxin17, SNAP29, and VAMP8 proteins were washed with lysis buffer five times and then collected by brief cDNA. This work was supported by grants from the National Key R&D centrifugation. Subsequently, the precipitated proteins were resolved by Program of China (2016YFA0501903), National Natural Science Foundation SDS/PAGE and detected by immunoblotting using a chemical luminescence- of China (31470749, 21621002, 91753113, 21822705), Science and Technol- based detection method. ogy Commission of Shanghai Municipality (17JC1405200), Strategic Priority Research Program of the Chinese Academy of Sciences (XDB20000000), and Circular Dichroism Spectroscopy. CD measurements were performed using a the start-up fund from the State Key Laboratory of Bioorganic and Natural Chirascan instrument (Applied Photophysics). A Hellma quartz cuvette with a Products Chemistry and Chinese Academy of Sciences.

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