Structural basis for the binding of SNAREs to the multisubunit tethering complex Dsl1
2020; Elsevier BV; Volume: 295; Issue: 30 Linguagem: Inglês
10.1074/jbc.ra120.013654
ISSN1083-351X
AutoresSophie M. Travis, Kevin DAmico, I-Mei Yu, Conor McMahon, Safraz A. Hamid, Gabriel Ramirez-Arellano, Philip D. Jeffrey, Frederick M. Hughson,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoMultisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–mediated membrane fusion in all eukaryotes. MTCs are thought to organize membrane trafficking by mediating the initial long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the yeast Dsl1 complex, the simplest known MTC, which is essential for coat protein I (COPI) mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggests how the Dsl1 complex might tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other to ER-associated SNAREs. Here, we used X-ray crystallography to investigate these Dsl1–SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. We found that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations, with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, will depend on the relative orientation of the two Dsl1 legs. These results underscore the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery. Multisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–mediated membrane fusion in all eukaryotes. MTCs are thought to organize membrane trafficking by mediating the initial long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the yeast Dsl1 complex, the simplest known MTC, which is essential for coat protein I (COPI) mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggests how the Dsl1 complex might tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other to ER-associated SNAREs. Here, we used X-ray crystallography to investigate these Dsl1–SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. We found that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations, with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, will depend on the relative orientation of the two Dsl1 legs. These results underscore the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery. Securing SNAREs for assemblyJournal of Biological ChemistryVol. 295Issue 30PreviewSNARE proteins are essential for exocytosis, mediating the fusion of vesicles with their target membrane. Tethering factors play a key role in chaperoning SNARE assembly; however, the underlying molecular mechanisms are not well-understood. Here, Travis et al. report two crystal structures of a yeast tethering factor, the Dsl1 complex, bound with two SNARE proteins, revealing new insights into how tethering factors bridge vesicles to target membranes, recruit multiple SNARE proteins, trigger their conformational changes, and facilitate SNARE assembly. Full-Text PDF Open Access Eukaryotic cells use vesicles to transport cargo between organelles and to the plasma membrane for exocytosis. These transport vesicles bear tail-anchored SNARE proteins that, in concert with complementary SNAREs in the target membrane, draw the two membranes into close apposition and facilitate membrane fusion. Each SNARE contains at least one SNARE motif, and sequence features within these motifs define four groups of SNAREs: Qa, Qb, Qc, and R (1Fasshauer D. Sutton R.B. Brunger A.T. Jahn R. 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Only a few such structures have previously been reported, and in no case has it been possible to integrate them into a complete structure of the MTC (6Fridmann-Sirkis Y. Kent H.M. Lewis M.J. Evans P.R. Pelham H.R. Structural analysis of the interaction between the SNARE Tlg1 and Vps51.Traffic. 2006; 7 (16420526): 182-19010.1111/j.1600-0854.2005.00374.xCrossref PubMed Scopus (43) Google Scholar, 12Abascal-Palacios G. Schindler C. Rojas A.L. Bonifacino J.S. Hierro A. Structural basis for the interaction of the Golgi-associated retrograde protein complex with the t-SNARE syntaxin 6.Structure. 2013; 21 (23932592): 1698-170610.1016/j.str.2013.06.025Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 22Yue P. Zhang Y. Mei K. Wang S. Lesigang J. Zhu Y. Dong G. Guo W. Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion.Nat. Commun. 2017; 8 (28112172): 1423610.1038/ncomms14236Crossref PubMed Scopus (50) Google Scholar, 25Baker R.W. Jeffrey P.D. Zick M. 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Of the five CATCHR-family MTCs, the Dsl1 complex is the smallest (∼250 kDa in Saccharomyces cerevisiae), the simplest (just three subunits), and the only one for which an essentially complete high-resolution structural model (based on overlapping crystal structures at resolutions ranging from 1.9 to 3.0 Å) is available (20Ren Y. Yip C.K. Tripathi A. Huie D. Jeffrey P.D. Walz T. Hughson F.M. A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1.Cell. 2009; 139 (20005805): 1119-112910.1016/j.cell.2009.11.002Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 32Tripathi A. Ren Y. Jeffrey P.D. Hughson F.M. Structural characterization of Tip20p and Dsl1p, subunits of the Dsl1p vesicle tethering complex.Nat. Struct. Mol. Biol. 2009; 16 (19151722): 114-12310.1038/nsmb.1548Crossref PubMed Scopus (87) Google Scholar). The central subunit, Dsl1, bridges the other two subunits, Tip20 and Sec39, which each form an elongated leg. These legs, because of a flexible hinge within the Dsl1 subunit, are able to adopt a broad range of relative orientations (20Ren Y. Yip C.K. Tripathi A. Huie D. Jeffrey P.D. Walz T. Hughson F.M. A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1.Cell. 2009; 139 (20005805): 1119-112910.1016/j.cell.2009.11.002Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). A distal portion of the Tip20 leg binds to the ER Qb-SNARE Sec20, whereas a distal portion of the Sec39 leg binds to the ER Qc-SNARE Use1 (20Ren Y. Yip C.K. Tripathi A. Huie D. Jeffrey P.D. Walz T. Hughson F.M. A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1.Cell. 2009; 139 (20005805): 1119-112910.1016/j.cell.2009.11.002Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 32Tripathi A. Ren Y. Jeffrey P.D. Hughson F.M. 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Fairman R. Hughson F.M. Roles of singleton tryptophan motifs in COPI coat stability and vesicle tethering.Proc. Natl. Acad. Sci. U.S.A. 2019; 116 (31712447): 24031-2404010.1073/pnas.1909697116Crossref PubMed Scopus (4) Google Scholar). Here, we have used structural and biochemical experiments to reveal the molecular nature of the interactions between the two legs of the Dsl1 complex and the ER SNAREs Sec20 and Use1. We find that each interaction involves a trihelical region of the corresponding SNARE. The observed binding modes would prevent each SNARE from adopting the closed conformation that has been observed for Qa-SNAREs, thereby leaving its SNARE motif free to engage other SNAREs. Placed in the context of the intact Dsl1 complex, the structures we report are consistent with the previous observations that the Dsl1 complex accelerates SNARE complex assembly, albeit modestly, and can bind fully assembled SNARE complexes (20Ren Y. Yip C.K. Tripathi A. Huie D. Jeffrey P.D. Walz T. Hughson F.M. A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1.Cell. 2009; 139 (20005805): 1119-112910.1016/j.cell.2009.11.002Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). More generally, our results highlight the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery. The S. cerevisiae Dsl1 complex subunit Tip20 was discovered as a cytoplasmic protein that interacts with the cytoplasmic domain of the ER Qb-SNARE Sec20 (40Sweet D.J. Pelham H.R. The TIP1 gene of Saccharomyces cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the cytoplasmic domain of Sec20p.EMBO J. 1993; 12 (8334998): 2831-284010.1002/j.1460-2075.1993.tb05944.xCrossref PubMed Scopus (47) Google Scholar). Biochemical experiments established that Tip20 binds an N-terminal region (residues 1–175), but not the SNARE motif, of Sec20 (20Ren Y. Yip C.K. Tripathi A. Huie D. Jeffrey P.D. Walz T. Hughson F.M. A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1.Cell. 2009; 139 (20005805): 1119-112910.1016/j.cell.2009.11.002Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Conversely, the N-terminal region of Tip20 (residues 1–81), which mediates binding to Dsl1, was not needed for binding to Sec20 (32). Therefore, we initially conducted crystallization screens using both full-length S. cerevisiae Tip20 and an N-terminally truncated version in combination with various constructs representing the N-terminal region of Sec20. Although a number of crystals were obtained, none of them diffracted well enough to allow structure determination. As an alternative approach, we screened orthologous Tip20•Sec20 complexes from other yeasts, co-expressed in bacteria. For this screen we used full-length Tip20 and an N-terminal fragment of Sec20 terminating just before the SNARE motif (Sec20NTD). Although most of the Tip20•Sec20NTD complexes were stable and soluble, only the Eremothecium gossypii complex yielded crystals. These initial crystals diffracted poorly, but replacing full-length Tip20 with an N-terminally truncated variant (Tip20A–E; Fig. 1A) led to the discovery of an additional crystal form that diffracted X-rays to 3.2 Å resolution. E. gossypii Tip20A–E•Sec20NTD was phased by molecular replacement using the previously reported S. cerevisiae Tip20 structure as a search model (32Tripathi A. Ren Y. Jeffrey P.D. Hughson F.M. Structural characterization of Tip20p and Dsl1p, subunits of the Dsl1p vesicle tethering complex.Nat. Struct. Mol. Biol. 2009; 16 (19151722): 114-12310.1038/nsmb.1548Crossref PubMed Scopus (87) Google Scholar). In the resulting structure (Fig. 1A and Table S1), E. gossypii Tip20A–E displays the characteristic hooked structure that appears to set Tip20 apart from other CATCHR-fold proteins (32Tripathi A. Ren Y. Jeffrey P.D. Hughson F.M. Structural characterization of Tip20p and Dsl1p, subunits of the Dsl1p vesicle tethering complex.Nat. Struct. Mol. Biol. 2009; 16 (19151722): 114-12310.1038/nsmb.1548Crossref PubMed Scopus (87) Google Scholar) (Fig. S1A). The greatest difference between E. gossypii Tip20 (in complex with Sec20NTD) and S. cerevisiae Tip20 (uncomplexed) lies in the angle between domains B and C (Fig. S1A). Although this might reflect an inherent difference between the two orthologues, it could also reflect intrinsic flexibility at the B/C domain junction. A comparable degree of flexibility near the B/C junction was also observed for the exocyst subunit Exo70 (41Hamburger Z.A. Hamburger A.E. West Jr., A.P. Weis W.I. Crystal structure of the S. cerevisiae exocyst component Exo70p.J. Mol. 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