Prefusion Structure of Syntaxin-1A Suggests Pathway for Folding Into Neuronal Trans-SNARE Complex Fusion Intermediate

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 solution 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) microscopy 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. A one di- Total RDC restraints 90 mensional [15N,1H]-TROSY for rotational correlation times Structure statistics (1D-TRACT) NMR experiment (22) indicated that this protein Violations (mean and s.d.)* consisted of at least two domains with significantly different ro- NOE constraints (Å) 0.033 ± 0.002 tational correlation times (Fig. S2). The observed relaxation PRE constraints (Å) 0.022 ± 0.002 decays could not be fitted with a single exponential, but only with Dihedral angle constraints (°) 0.692 ± 0.039 two (or more) exponentials. Multiple rotational components RDC constraints (Hz) 0.132 ± 0.013 likely arise from distinct domains with different dynamic Deviations from idealized geometry ± behaviors connected by flexible linkers, perhaps reflecting the Bond lengths (Å) 0.002 0.000 ± “open/closed” equilibrium of the Habc and H3 domains. Be- Bond angles (°) 0.386 0.005 Impropers (°) 0.306 ± 0.008 cause of these complications, we decided to focus our structural † study on the SNARE motif in a membrane-mimetic environment. Average pairwise rmsd (Å) Backbone (residues 189–286) 1.57 ± 0.42 – ± Syntaxin (Residues 183–288) in DPC Micelles. In this H3 plus TM Heavy (residues 189 286) 2.56 0.37 – ± domain syx construct (residues 183–288), all except the first few Backbone (H3N, residues 197 224) 0.51 0.26 – ± N-terminal residues exhibited chemical shifts and secondary Heavy (H3N, residues 197 224) 1.66 0.28 – ± chemical shift patterns similar to those in the full-length syx, Backbone (H3C, residues 227 251) 0.89 0.42 – ± indicating very similar structures of the corresponding domains Heavy (H3C, residues 227 251) 1.86 0.32 – ± (Fig. S3). The 1D-TRACT experiment indicated a single rota- Backbone (HTM, residues 261 282) 0.44 0.21 Heavy (HTM, residues 261–282) 1.37 ± 0.28 tional correlation time (τc) of 10 ns (Fig. 1B), which is consistent 15 with the NMR N-R1 and R2 relaxation measurements (Fig. *No NOE and PRE violations are greater than 0.5 Å, no dihedral angle vio- S4), in which the average of isotropic rotational correlation times lations are more than 5°, and the maximum RDC violation is 0.686 Hz. † was 10.4 ± 0.3 ns if all residues were used, or 12.1 ± 0.3 ns if only Calculated for 20 lowest energy conformers.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1314699110 Liang et al. Downloaded by guest on September 26, 2021 indicated by size-exclusion chromatography (Fig. 1A). We The third helix is the transmembrane helix (HTM), located be- conclude that a single syx molecule (12.3 kDa) is present in tween H3N and H3C in the projection shown in Fig. 2C. Its helical each micelle (the size of a protein-free DPC micelle is around axis forms a 70° angle against the plane defined by H3N and H3C 25 kDa). The monomeric form of syx was further confirmed by (Fig. 2D). There is an additional short, relatively flexible helix EPR measurements of spin-labeled single cysteine mutants, as comprising residues 190–194. The remaining N- and C-terminal the EPR spectra exhibit sharp lines with no indication of the line residues, as well as the juxtamembrane linker region (residues broadening that would be expected from a spin-spin interaction 252–260) between H3C and HTM, are disordered. of oligomeric species (Fig. 1D). The EPR line shapes remained the same when the spin-labeled sample was 20-fold diluted with Interaction of SNAP-25 and Munc18 with Micelle-Bound Syntaxin. To unlabeled protein, supporting the monomeric state of syx in DPC examine how SN25 and Munc18 interact with micelle-bound syx, micelles. Importantly, our syx construct in DPC micelles was we collected heteronuclear single quantum correlation (HSQC) biochemically fully functional, as shown by its ability to form spectra of syx (residues 183–288) in DPC and mixed them with native heterotrimeric SNARE complex (Fig. 1C). When the syx/ equal molar amounts of SN25 or Munc18. On addition of SN25, DPC NMR sample was used to form an acceptor complex with very small chemical shift changes were observed, indicating that SN25 and a short syb peptide in DPC (10), this complex rapidly SN25 does not efficiently interact with micelle-bound syx (Fig. 3). and efficiently formed SDS-resistant SNARE complex after re- This result is not surprising because the formation of the binary action with soluble syb (residues 1–96). syx-SN25 complex is inefficient in membranes if not assisted by other proteins (9). In contrast, when Munc18 was added to DPC- Solution NMR Structure of Syx (residues 183–288). Syx-H3-TM in bound syx, many HSQC resonances showed significant chemical DPC produced excellent NMR spectra that could be fully shift changes. Most interestingly, all substantial chemical shift assigned (Fig. S3A). Structures were calculated based on chem- changes were localized in the H3N helix, whereas H3C and HTM ical shift, J-coupling, nuclear Overhauser effect (NOE), para- residues showed very little change on the addition of Munc18 magnetic relaxation enhancement (PRE), and residual dipolar (Fig. 3). The selective binding of Munc18 to H3N is reminiscent coupling (RDC) data, as described in SI Materials and Methods. of its interaction in the crystal structure of the Munc18-syx(1– NMR and refinement statistics are listed in Table 1. The struc- 267) complex (16) and supports the functional relevance of the ture is characterized by three well-ordered long α-helices (Fig. segmented SNARE motif structure of syx. SN25 binding to syx 2A), as indicated by secondary chemical shifts (Fig. S3B) and was not facilitated by Munc18 in this system. a range of NMR dynamics measurements (Fig. S4). The relative BIOPHYSICS AND

orientations of these three helices were defined by NMR PRE Positions of Helices in Membrane. The helical SNARE motif struc- COMPUTATIONAL BIOLOGY (Fig. S5) and RDC (Fig. S6) measurements. The three helices ture must be a result of the presence of the TM domain and as- range from residues 197–224 to 227–251 and 261–282, with sociated lipid micelle, as this motif is disordered in soluble syx backbone rmsds of 0.51 ± 0.26, 0.89 ± 0.42, and 0.44 ± 0.21, (15). When a sphere with a diameter of 35 Å is grafted onto the respectively (Fig. S7). The first helix (H3N) is highly curved, and syx structure to roughly represent the size of the hydrophobic core the angle between the helix axes defined by the first two and last of a DPC micelle (23), H3N nicely follows the curved micelle– two helical turns is ∼115° (Fig. 2C). The second helix (H3C)is water interface (Fig. 2F). However, the straight H3C appears to relatively straight. Residues 225 and 226 form a turn, which transsect the hydrophobic core of the micelle at a shallow depth. It orients the connecting ends of H3N and H3C at an ∼95° angle. is possible that in a more planar lipid bilayer environment, H3N,

Fig. 2. Solution NMR structure of syntaxin-1A in dodecylphosphocholine micelles. (A) Domain structure of monomeric syx (residues 183–288) in DPC micelles derived in this study compared with domain structures of previous high-resolution structures of syntaxin (6, 16, 20). Helical domains are indicated as boxes, and irregular structures as lines. (B) Ensemble representation of 20 structures of syx (residues 183– 288) with the lowest violation energies in DPC micelles. (C) Ribbon diagram of a representative structure of syx (residues 183–288) in DPC micelles. (D–F) Three different views of the structure ensemble grafted onto a 35-Å sphere representing the hydrophobic core of a DPC micelle; only residues 189–286 are displayed. The long helices are colored in red (residues 197–224), purple (residues 227–251), and gold (residues 261–282), respectively.

Liang et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 0.14 SNAP25 determine molecular orientations and distances in membranes (24-26). Very briefly, fluorophores are attached at different 0.12 Munc18 positions in the molecule, and their distances from the mem- fl 0.10 brane surface are measured by uorescence interferometry to a precision of about ±5 Å in the most favorable cases (Figs. 4 A– (ppm) 0.08 C). To check whether the H3N and H3C helices of prefusion syx were lying in the plane of the membrane, we labeled single Cys comp 0.06 mutants of syx at residues 192 and 225 (i.e., the N-termini of H

Δδ 3N 0.04 and H3C) with Alexa546 and found that both sites were located ± ± 0.02 at or very near the membrane surface (1.4 0.5 and 0.8 0.2 nm, respectively) (Fig. 4 D and E; see also Fig. 5A, Left). 0.00 Even though the two SNARE motif helices clearly lie at the 200 220 240 260 280 membrane surface, the angle between them may not be the same Residue Number as in the DPC micelle. In fact, the joints between the three he- fl Fig. 3. Chemical shift changes after binding equimolar amounts of un- lices are the most exible parts of the molecule (Fig. S4). When labeled SNAP-25 or Munc18 to 13C, 15N-labeled syntaxin-1A. Compound we attempted to assemble SNARE complexes from this state at 2 2 1/2 chemical shift changes, defined as Δδcomp = [ΔδHN + (ΔδN/6.5) ] (43), are the membrane surface, very little distance change was observed plotted versus residue numbers of syx(183–288). (2.2 ± 0.3 and 1.2 ± 0.4 nm, respectively; Fig. 4H) after addition of SN25 with or without soluble syb (residues 1–96). This result is not surprising because it has been previously reported that in similar to H3C, might adopt a straighter conformation, and that vitro assembly of isolated SNAREs at membrane surfaces is not fi the plane de ned by H3N and H3C would likely coincide with the efficient and is very slow (4, 9). As is customary in all recon- – bilayer water interface. In addition, HTM might adopt a different stituted SNARE fusion assays, we next used a target membrane angle relative to the H3N and H3C plane, as the juxtamembrane acceptor SNARE complex, which was preassembled from syx linker is quite flexible in our detergent-derived structure. NOEs and SN25 in detergent before reconstitution into lipid bilayers. between backbone amide protons and lipid acyl chain or water Our version of this acceptor SNARE complex also included the protons (Fig. S8)confirmed that most H3N and H3C residues were small C-terminal peptide from syb (residues 49–96), which pre- – at the lipid water interface, whereas HTM was deeply embedded in vents the formation of the competing nonproductive syx2:SN25 the hydrophobic core of the DPC micelle. complex (10) (Fig. 1C). When this acceptor complex was reconstituted into lipid bilayers, the labeled residues 192 and 225 FLIC Measurements in Lipid Bilayers. To examine whether syx were 4.7 ± 0.3 and 4.1 ± 0.3 nm away from the membrane, re- positions in a lipid bilayer, as predicted from the DPC structure, spectively (Figs. 4 F and G). When the acceptor complex was and to determine how the orientations of the H3 helices might subsequently incubated with the soluble syb, the distance of the change during SNARE complex assembly, we used FLIC mi- H3N marker at position 192 was increased to 6.0 ± 0.3 nm from croscopy, which has become a well-established technique to the membrane surface, but the H3C marker at position 225 did

ABC Fig. 4. Distance measurements by FLIC microscopy. 1 (A) Principle of distance measurements by FLIC microscopy. Fluorescently labeled proteins are reconstituted into planar membranes supported on a 225 192 patterned silicon chip with microscopic steps of sil- 225 192 icon dioxide. The fluorescence intensity depends on 225 192 the position of the fluorophore with respect to the 225 192 standing modes of the exciting (green) and emitting light (red) in front of the reflecting silicon surface. SiO2 norm. Fluorescence 0 40μm The position is determined by the variable height of 0 200 400 Si the oxide steps and the constant mean distance be- Oxide Thickness [nm] tween silicon oxide and labeled residues. (B)Example D E H of a FLIC image. Syx was labeled at residue 192 and 300 syx syx reconstituted into a supported membrane. A set of 16 @192 @225 different oxide terraces is marked with a red square 6 covering an area of 40 × 40 μm2.(C)Atypicalsetof fluorescence intensity versus oxide thickness extrac- count ted from 16 different oxide levels in (B). Also shown is 0 4 the best fitoftheFLICtheory,whichresultedin a fluorophore-membrane distance d = 0.8 nm. (D– 0 10 0 10 m G) Histograms of fluorophore to membrane distances dm [nm] dm [nm] [nm] ∼ m 2 dm obtained from 1,000 data sets, as shown in (C), F d with syx labeled with Alexa 546 at residues 192 (D) G and 225 (E), and the acceptor complex (ΔN) labeled 300 ΔN ΔN at syx residues 192 (F)and225(G). Red curves are @192 @225 0 Gaussian fits to the histograms to confirm a normal distribution of the data. (H) Mean distances of resi- +syb +syb +syb +syb +SN25 +SN25 count -2 dues 192 and 225 of syx and the acceptor complex from the membrane, as determined by FLIC before 0 syx syx acceptor acceptor (black) and after (blue) binding of soluble syb (resi- – 0 10 0 10 @192 @225 complex complex dues 1 96) from 6 to 8 independent experiments. In dm [nm] dm [nm] @192 @225 the case of syx, soluble syb was added in the presence of SN25. Error bars represent SEMs.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1314699110 Liang et al. Downloaded by guest on September 26, 2021 Discussion Segmented Helical Structure of Prefusion Syntaxin. The most strik- ing feature of the prefusion syx structure is that the SNARE motif is neither flexible, as in soluble syx (15, 16), nor a continuous helix, as in the postfusion SNARE complex (20). The SNARE motif forms two long helices (H3N and H3C) connected by a turn at residues Ser225 and Gln226. The latter is the single Gln residue in the heptad repeat of syx and forms the “0-layer” in the SNARE complex. This highly conserved 0-layer comprises an Arg from syb and one plus two Glns from syx and SN25, respectively. It ensures proper register of the hydrophobic heptad repeat interactions in the coiled-coil complex (19). A previous study of membrane-bound prefusion syb also found that its N-terminal helix is terminated just two residues before the 0-layer Arg (27). The observation that the N-terminal SNARE motifs of syx and syb have a high propensity for helix formation even in the open prefusion state suggests that these might form nucleation sites for SNARE folding and assembly, and the termination of these helices at the 0-layer strongly supports the notion that this structural feature may act as a proofreading site for SNARE complex formation (28). On the basis of the observed folding of the individual SNAREs, it is likely that a partially zippered SNARE assembly intermediate with exposed 0-layer residues may persist for a sufficient amount of time to fulfill the proposed proofreading function. It is possible, or perhaps even likely, that an arrested partially folded trans-SNARE complex that is zippered up to this Fig. 5. Molecular models of syntaxin-1A structure in lipid bilayers (A) and its layer may wait at this stage for a trigger to fully zipper through the possible progression to engage in trans and cis-SNARE complexes at the site end of the SNARE sequences, and thereby complete fusion. This BIOPHYSICS AND of fusion (B). (A) Left to right, the conformation of prefusion syx as de- COMPUTATIONAL BIOLOGY termined by NMR in lipid micelles and validated by FLIC microscopy in lipid trigger may be provided by complexin (29-30), synaptotagmin (31- bilayers, a model of the ΔN acceptor SNARE complex as determined by FLIC 32), calcium, special lipids, or a combination. Similar conclusions microscopy, and a model of the postfusion SNARE complex after reacting the were reached by a single-molecule study on SNARE complex folding ΔN acceptor complex with syb (residues 1–96) as determined by X-ray crys- (33), but the current work, together with a similar study on syb (27), tallography and FLIC microscopy. (B) The incorporation of these findings into adds high-resolution structural evidence for this behavior. a possible model of SNARE assembly and folding promoting fusion. From left to right are depicted the prefusion structures of syx (this work and ref 6), syb SNARE Assembly and Membrane Fusion. Our result, that open syx (27), and SN25 (unknown), and then a model of the trans-SNARE complex without the regulatory Habc and N-peptide domains associates fi based on this work and refs. 20 and 27, and nally the postfusion cis-SNARE with the membrane surface, explains why this molecule cannot complex (20). The angles between the different elements of secondary easily form a SNARE complex in membranes. However, when structure may be partially flexible. a preassembled acceptor complex is reconstituted in lipid model membranes, it stands above the membrane surface and is ready not move by a statistically significant distance; that is, it remained to accept the cognate vesicle SNARE synaptobrevin for SNARE fi at 4.3 ± 0.2 nm from the membrane surface (Fig. 4H). assembly, folding, and fusion (Fig. 5B). Although the speci c Apparently, the acceptor complex adopted a “flag–pole” con- acceptor complex that was used here and in previous work (9-10, 34) does not exist in vivo, alternate proteins, including syntaxin’s formation in which H3C was perpendicular to the membrane own Habc domain and Munc18, likely fulfill a similar function surface, whereas the H3N “flag” was flexibly or rigidly attached to the top of the “pole” so that the average distance of residue 192 and prepare syntaxin in an upright conformation with a tran- from the membrane surface was only a little more than that of siently open binding site, ready to accept synaptobrevin on residue 225 at the top of the pole (Fig. 5A, Middle). Completing incoming vesicles and ready to promote fusion between the SNARE complex assembly with soluble syb moves the flag resi- two membranes. due 192 up to 6.0 nm, about 2 nm less than expected from the The interaction between Munc18 and micelle-bound syx sug- crystal structure of the postfusion SNARE complex (Fig. 5A, gests that the excitatory effect of Munc18 in SNARE-mediated Right) and the orientation of SNARE proteins within the sup- exocytosis may involve its preferential binding to the H3N helix of ported membrane (Fig. S9). The postfusion SNARE complex is syx and that the elusive trans-SNARE complex may involve a straight helical bundle, perpendicular to the membrane surface segmented helical elements around H3N and H3C (Fig. 5B, Mid- (20, 25), and residues 192 and 225 are expected to be 10 and dle). The results of this work and recent competitive binding and 5 nm away from the membrane surface, respectively. FLIC site-directed spin-labeling studies (12, 18, 35) clearly show that the membrane is an important factor in keeping the SNARE motif of microscopy determines the average distance of all molecules, of syx in a prefolded helical state and that proteins similar to the which 87% are oriented with their cytoplasmic domains facing Habc domain (14), Munc18 (36), and Munc13 (37) are required away from the substrate (Fig. S9). Taking this into account and to shuttle this form of the SNARE motif from the membrane assuming that all acceptor complexes on the proximal side of the surface to the nascent SNARE complex. membrane react with syb, we would expect to measure a distance of 7.9 nm from the membrane surface for residue 192. The result Materials and Methods of 6.0 nm means that in our experiment, not all acceptor com- SNARE proteins were all expressed in BL21(DE3) cells under the control of the plexes reacted with syb; the postfusion SNARE complexes can fi α = T7 promoter (pET28a), puri ed and reconstituted in DPC as described pre- wobble in a cone of 39° from the membrane normal, which in viously (38). Typical NMR samples contained between 0.2 and 1 mM protein part may be because only the transmembrane helix from syntaxin and 100 and150 mM DPC in 10 mM Hepes, 10 mM MES, 10 mM acetate at pH is present in our case; or a combination of the two. 5.5 buffer with 150 mM NaCl, 5 mM DTT, and 1 mM EDTA. For samples used

Liang et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 for NOESY experiments, a phosphate buffer and D38-DPC were used to transfer, and the second lipid leaflet containing Alexa labeled syx was formed minimize NOE transfer, and for spin-labeled samples, DTT was not present. by the addition of syx-containing proteoliposomes. Average distances of the NMR experiments were typically carried out at 40 °C. fluorescent dyes from a reflective interface were extracted from the FLIC For structure calculation, dihedral angle restraints were predicted from images. For each membrane condition, all fit results were pooled to yield 800– + the TALOS program (39) on the basis of experimental chemical shifts. NOE 1,200 distances with normal Gaussian distribution profiles. Details about all 15 13 distances were extracted and calibrated from N-edited and C-edited these methods are provided in SI Materials and Methods. NOESY spectra automatically with the PASD routine (40) in Xplor-NIH (41) and were further confirmed manually. Long-range PRE distances were Data Depositions. NMR chemical shifts and other data have been deposited in converted from peak intensity ratios of paramagnetically and diamagneti- the Biomolecular Magnetic Resonance Data Bank, and the structural coor- cally labeled samples. RDCs providing orientational information were mea- dinates of syx (residues 183–288) have been deposited in the Protein Data sured in a compressed charged gel environment. In addition, intrahelical Bank under accession number 2M8R. hydrogen bonds were used on the basis of a range of NMR dynamics measurements, as well as NOE and RDC patterns. The final 20 representative structures were selected on the basis of the lowest total violation energies. ACKNOWLEDGMENTS. We thank Dr. P. Fromherz, Dr. A. Lambacher, and Mr. H. Vogl (Max-Planck-Institute for Biochemistry, Martinsried, Germany) Ramachandran plot statistics as calculated using PROCHECK-NMR (42) are for their help with the manufacturing of the FLIC chips. We also thank most favored (80.0%), additionally allowed (14.6%), generously allowed Dr. Reinhard Jahn for providing plasmids of SNARE proteins and Damian (4.3%), and disallowed (1.1%). Dawidowski and Dr. David Cafiso for providing plasmids of three single FLIC substrates with 16 levels of oxide thickness ranging from 10 to 450 nm cysteine syx mutants and access to their EPR equipment. This work has been were used. A lipid monolayer was first prepared by Langmuir-Blodgett supported by National Institutes of Health Grant P01 GM072694.

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