Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate Binyong Liang, Volker Kiessling, and Lukas K. Tamm1 Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908 Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved October 15, 2013 (received for review August 5, 2013) The assembly of the three neuronal soluble N-ethylmaleimide- and H3 domains of syx are in dynamic equilibrium between sensitive factor attachment protein (SNAP) receptor (SNARE) “closed” and “open” conformations, which is regulated by proteins synaptobrevin 2, syntaxin-1A, and SNAP-25 is the key Munc18-1 and Munc13, and which thereby controls synaptic step that leads to exocytotic fusion of synaptic vesicles. In the fully vesicle fusion (11–14). Only the open, not the closed, confor- assembled SNARE complex, these three proteins form a coiled-coil mation promotes formation of the SNARE complex, and thus four-helix bundle structure by interaction of their respective membrane fusion. SNARE motifs. Although biochemical and mutational analyses Although the three-helix Habc domain is well-ordered, the strongly suggest that the heptad-repeat SNARE motifs zipper H3 domain is flexible in a soluble syx construct without its TM into the final structure, little is known about the prefusion state domain (15). Two different modes of binding of Munc18-1 to of individual membrane-bound SNAREs and how they change soluble syx have been observed. In a crystal structure of the syx- conformation from the unzippered prefusion to the zippered Munc18 complex, Munc18 locks syx in a “closed” conformation. postfusion state in a membrane environment. We have solved the The N-terminal half of the H3 domain forms an interrupted helix solution NMR structure of micelle-bound syntaxin-1A in its pre- that is stabilized by interaction with Habc and Munc18 (16). fusion conformation. In addition to the transmembrane helix, the Alternatively, Munc18 can just bind to the N-terminal peptide of SNARE motif consists of two well-ordered, membrane-bound syx (13, 17). Interestingly, the Munc18-regulated open/closed BIOPHYSICS AND helices separated by the “0-layer” residue Gln226. This unexpected equilibrium of syx is shifted toward being more open in the COMPUTATIONAL BIOLOGY structural order of the N- and C-terminal halves of the uncom- membrane-bound and more closed in the soluble form of syx plexed SNARE motif suggests the formation of partially zippered (18). In the soluble and the detergent-bound postfusion SNARE SNARE complex intermediates, with the 0-layer serving as a proof- complex, the SNARE motifs form uninterrupted helices (19, 20). reading site for correct SNARE assembly. Interferometric fluores- Moreover, the SNARE helices continue through the juxta- cence measurements in lipid bilayers confirm that the open membrane linker regions to the TM domains in the latter struc- SNARE motif helices of syntaxin interact with lipid bilayers and ture (20). Because some structures and conformational equilibria that association with the other target-membrane SNARE SNAP- are different for membrane-bound than for soluble SNAREs (18, 25 lifts the SNARE motif off the membrane as a critical prerequisite for SNARE complex assembly and membrane fusion. Significance membrane protein structure | nuclear magnetic resonance | FLIC microscopy Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs) are the key molecules that control fi eurotransmitter release in eukaryotic cells requires the fu- fusion in intracellular vesicle traf c. A special case of vesicle-to- Nsion of synaptic vesicles with the presynaptic plasma mem- plasma membrane fusion is exocytosis of synaptic vesicles at brane. This fast and precise process is mediated by many proteins the presynaptic membrane to release neurotransmitters into (1–3). The minimal neuronal exocytotic fusion machinery includes the synaptic cleft. Structures are known of postfusion SNARE three soluble N-ethylmaleimide-sensitive (NSF) factor attachment complexes, including a famous four-helix bundle with parallel protein receptor (SNARE) proteins: synaptobrevin-2 (syb) in the C-terminal transmembrane domains. However, prefusion struc- trans vesicle membrane and syntaxin-1A (syx) and SNAP-25 (SN25) in tures and the structures of intermediate -SNARE com- the plasma membrane (4). These proteins possess either one or plexes remain much more elusive. Using nuclear magnetic two conserved SNARE motifs of 60–70 amino acids, which on resonance, we have determined the prefusion structure of the SNARE assembly are capable of forming a four-stranded coiled- lipid-bound t-SNARE syntaxin-1A, with its transmembrane do- fi coil helical bundle (5). Folding, assumed to occur by N- to C- main, and con rmed its lipid interactions and conformational terminal zippering of these helices, is thought to be sufficient to transitions on co-t-SNARE SNAP-25 binding by high-resolution merge the two opposing membranes. However, little is known interference contrast microscopy in lipid bilayers. We discuss about how the individual SNARE proteins change their con- how these structures and their folding into later SNARE com- formations from the unzippered prefusion to the zippered plexes might drive membrane fusion. postfusion states in membranes. Whereas SN25 and syb Author contributions: B.L., V.K., and L.K.T. designed research; B.L. and V.K. performed mostly consist of the SNARE motifs and, in the case of syb, research; B.L., V.K., and L.K.T. analyzed data; and B.L., V.K., and L.K.T. wrote the paper. a transmembrane (TM) domain, syx possesses two regulatory The authors declare no conflict of interest. domains, a three-helix bundle (Habc) domain (6, 7) and a short N-terminal peptide (N-peptide) (8), in addition to its SNARE This article is a PNAS Direct Submission. motif (H3 domain) and TM domain. Although neither of these Database deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2M8R). NMR chemical shifts and other data have been additional domains are required in vitro for fast fusion per se (9, deposited in the Biomolecular Magnetic Resonance Data Bank, http://deposit.bmrb.wisc. 10), the interaction between these domains and the SNARE edu (accession no. 19266). motif in the presence of Munc18-1, Munc13, and other regula- 1To whom correspondence should be addressed. E-mail: [email protected]. “ ” tory proteins is required upstream to precondition the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. SNAREs for efficient assembly and subsequent fusion. The Habc 1073/pnas.1314699110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1314699110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 AB 1.4 Protein 1.0 800 Standard (kDa) 158 17 Syx in DPC 0.8 Rα = 21 Hz 600 670 Syx in CHAPS Rβ = 46 Hz 0.6 44 Fig. 1. syntaxin-1A in dodecylphosphocholine 400 Solvent micelles is monomeric and capable of forming SNARE 0.4 complex. (A) Size-exclusion chromatogram shows UV280 (mAU) 200 Relative Intensity that syx (residues 183–288) is monomeric in DPC 0.2 micelles. For comparison, it forms higher-order 0 oligomers in CHAPS micelles, which is standard in 0.0 some commonly used reconstitution protocols. (B)A 0 5 10 15 20 25 0.05 0.10 0.15 0.20 0.25 0.30 Elution Volume (ml) t (s) 1D-TRACT NMR experiment determines the overall rotational correlation time of the syx (residues 183– CD SN25 Syx Syb49 Syb96 ΔN ΔN+Syb96 std 200 μM spin- t(min) 0.5 1 2 5 30 +boil labeled sample 288)/DPC complex to be 10 ns. (C) A SDS-resistant kDa fi 250 SNARE complex forms quickly and ef ciently when 10 μM spin-labeled 150 Δ 100 sample mixed wth the acceptor complex ( N complex) in DPC is reac- 75 190 μM WT-syx – ted with soluble syb (residues 1 96). Characteristic 50 SNARE complex bands appear and the SNAP-25 37 band at 25 kDa disappears within 30s of reaction 25 time. (D) Representative EPR spectra of nitroxide- 20 labeled single-cysteine (S225C) mutant of syx in DPC 15 micelles at both high and low spin concentrations 10 5 show no dipolar spin-spin interaction, indicating that syntaxin-1A is monomeric. 21), and as all previously reported high-resolution structures of ordered residues were included. On the basis of the rotational prefusion syx have been obtained for their soluble portions only correlation times, the size of the protein–detergent complex can (6, 15, 16), it is of great interest to elucidate the structure of be estimated to be 35–45 kDa, which is similar to the value prefusion syx in a membrane-mimetic environment and to demonstrate its relevance in lipid bilayers. Here, we used solu- tion NMR to study the structure and dynamics of syx in lipid Table 1. NMR and refinement statistics for syntaxin (183–288) micelles and fluorescence interference contrast (FLIC) micros- copy to validate its conformation and activity in lipid bilayers. NMR constraints Total unique NOE 1085 Results Intraresidue 512 Full-Length Syntaxin (Residues 1–288). We first attempted to solve Interresidue the structure of a full-length syx (residues 1–288) in dodecyl- Sequential (ji – jj = 1) 349 phosphocholine (DPC) micelles (Fig. S1A). More than two Medium-range (ji – jj <= 4) 218 thirds of all residues (197/288) could be assigned by standard Long-range (ji – jj ≥ 5) 6 heteronuclear resonance assignment strategies. The secondary PRE restraints 378 chemical shifts indicated that this protein was rich in α-helical Intrahelical hydrogen bonds 59 content (Fig. S1B). Compared with a previous NMR study of Total dihedral angle restraints 216 soluble syx (residues 26–230) (15), membrane-anchored syx TALOS ϕ 91 showed similar secondary structure for the Habc domain, but the TALOS ψ 91 3 N α SNARE domain exhibited a much more well-ordered α-helical J(H H )34 structure compared with its soluble counterpart.