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Decoding Three Distinct States of the Syntaxin17 SNARE Motif in Mediating Autophagosome–Lysosome Fusion

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Decoding Three Distinct States of the Syntaxin17 SNARE Motif in Mediating Autophagosome–Lysosome Fusion Decoding three distinct states of the Syntaxin17 SNARE motif in mediating autophagosome–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 protein receptor (SNARE) protein, can associate However, the detailed molecular mechanisms underlying the co- with ATG8 family proteins 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 autophagy 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 eukaryotes 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,
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