A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly
2004; Elsevier BV; Volume: 279; Issue: 9 Linguagem: Inglês
10.1074/jbc.m312064200
ISSN1083-351X
AutoresDirk Fasshauer, Martin Margittai,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoThe SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca2+-dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither N- nor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1:1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate. The SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca2+-dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither N- nor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1:1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate. Following a Ca2+ influx into the synaptic terminal, neurotransmitter is rapidly released from synaptic vesicles that fuse with the plasma membrane. The synaptic vesicle protein synaptobrevin 2 (also referred to as vesicle-associated membrane protein 2, or VAMP 2) and the plasma membrane proteins syntaxin 1a and SNAP-25 1The abbreviations used are: SNAP-25, soluble N-ethylmaleimide-sensitive factor attachment protein of 25 kDa; SNARE, N-ethylmaleimide-sensitive factor attachment protein receptor; FRET, fluorescence resonance energy transfer; OG, Oregon Green; TR, Texas Red; IAANS, anilinonaphthalenesulfonate iodoacetamide; PBS, phosphate-buffered saline; Syb, synaptobrevin 2; BONT/A and BONT/E, botulinum neurotoxin A and E, respectively; Syx, syntaxin. play a central role during this process. They belong to the family of so-called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are essential for all vesicular trafficking steps. It is believed that SNAREs generally initiate the process of membrane fusion by the sequential formation of a stable heteromeric "trans" complex between the vesicular and the target membranes. This basic SNARE machinery is thought to be tightly controlled by a complex protein network (for a review, see Refs. 1Chen Y.A. Scheller R.H. Nat. Rev. Mol. Cell Biol. 2001; 2: 98-106Crossref PubMed Scopus (878) Google Scholar, 2Rizo J. Sudhof T.C. Nat. Rev. Neurosci. 2002; 3: 641-653Crossref PubMed Scopus (436) Google Scholar, 3Jahn R. Lang T. Sudhof T.C. Cell. 2003; 112: 519-533Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar, 4Fasshauer D. Biochim. Biophys. Acta. 2003; 1641: 87-97Crossref PubMed Scopus (93) Google Scholar, 5Ungar D. Hughson F.M. Annu. Rev. Cell Dev. Biol. 2003; 19: 493-517Crossref PubMed Scopus (232) Google Scholar). Syntaxin and synaptobrevin each contain a single SNARE complex-forming coiled-coil region, termed the SNARE motif, directly adjacent to their C-terminal transmembrane domain. Syntaxin 1a contains an additional regulatory N-terminal region, called the Habc domain. SNAP-25 is composed of two SNARE motifs connected by a long linker and is attached to the membrane by palmitoyl modifications in this region. Upon SNARE complex formation, major conformational changes occur, with mostly unstructured proteins forming a stable α-helical complex (6Fasshauer D. Bruns D. Shen B. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 7: 4582-4590Abstract Full Text Full Text PDF Scopus (169) Google Scholar, 7Fasshauer D. Otto H. Eliason W.K. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 8Fasshauer D. Eliason W.K. Brünger A.T. Jahn R. Biochemistry. 1998; 37: 10354-10362Crossref PubMed Scopus (208) Google Scholar, 9Hazzard J. Sudhof T.C. Rizo J. J. Biomol. NMR. 1999; 14: 203-207Crossref PubMed Scopus (75) Google Scholar, 10Margittai M. Fasshauer D. Pabst S. Jahn R. Langen R. J. Biol. Chem. 2001; 276: 13169-13177Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The mechanical force of assembly could potentially be used to overcome the repulsive forces between membranes (7Fasshauer D. Otto H. Eliason W.K. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). The assembled core complex consists of an elongated four-helix bundle with synaptobrevin and syntaxin each contributing one helix and SNAP-25 contributing two helices (11Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar). In the interior, 16 layers of mostly hydrophobic amino acids are formed; however, an unusual hydrophilic layer of three glutamine residues and one arginine residue is located in the center of the bundle. Each helix can be assigned to a different SNARE subfamily. Accordingly, the SNARE helices contributing a glutamine to the zero-layer are named Qa- (syntaxin), Qb- (first half of SNAP-25; SN1), and Qc-SNARE (second half of SNAP-25; SN2), whereas synaptobrevin is the R-SNARE (12Fasshauer D. Sutton R.B. Brünger A.T. Jahn R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15781-15786Crossref PubMed Scopus (756) Google Scholar, 13Bock J.B. Matern H.T. Peden A.A. Scheller R.H. Nature. 2001; 409: 839-841Crossref PubMed Scopus (527) Google Scholar). A very similar structure was found in the late endosomal SNARE complex, which is only distantly related to the synaptic complex (14Antonin W. Fasshauer D. Becker S. Jahn R. Schneider T.R. Nat. Struct. Biol. 2002; 9: 107-111Crossref PubMed Scopus (210) Google Scholar). In both complexes, the SNARE helices are aligned in parallel, suggesting that assembly between vesicle and target membrane originates at the membrane-distal N-terminal ends of the respective SNARE motifs and proceeds in a zipper-like fashion toward the membrane anchors (15Hanson P.I. Heuser J.E. Jahn R. Curr. Opin. Neurobiol. 1997; 7: 310-315Crossref PubMed Scopus (334) Google Scholar, 16Hay J.C. Scheller R.H. Curr. Opin. Cell Biol. 1997; 9: 505-512Crossref PubMed Scopus (254) Google Scholar). That SNARE proteins indeed constitute a minimal membrane fusion machinery was inferred from experiments in which SNAREs were shown to slowly catalyze fusion between liposomes (17Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar). Since the two Q-SNAREs syntaxin and SNAP-25 mainly reside in the plasma membrane, they are presumed to cooperate in providing the binding site for the vesicular R-SNARE synaptobrevin. This concept was substantiated by the fact that both Q-SNAREs form a stable complex in vitro. A closer inspection of the syntaxin-SNAP-25 complex, however, revealed that it already consists of a similar four-helix bundle, in which the binding site for synaptobrevin is taken by a second syntaxin molecule (7Fasshauer D. Otto H. Eliason W.K. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 10Margittai M. Fasshauer D. Pabst S. Jahn R. Langen R. J. Biol. Chem. 2001; 276: 13169-13177Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Xiao W. Poirier M.A. Bennett M.K. Shin Y.K. Nat. Struct. Biol. 2001; 8: 308-311Crossref PubMed Scopus (91) Google Scholar, 19Zhang F. Chen Y. Kweon D.H. Kim C.S. Shin Y.K. J. Biol. Chem. 2002; 277: 24294-24298Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Although one cannot exclude the possibility that the second syntaxin is necessary to stabilize the Q-SNARE interaction, it seems likely that a 1:1 syntaxin-SNAP-25 interaction would provide a more straightforward synaptobrevin binding site in vivo. The latter scenario is corroborated by studies on a homologous Q-SNARE complex involved in exocytosis in yeast, for which a 1:1 interaction of Ssop (syntaxin homologue) and Sec9p (SNAP-25 homologue) is believed to provide the binding site for Sncp (synaptobrevin homologue) (20Rice L.M. Brennwald P. Brünger A.T. FEBS Lett. 1997; 415: 49-55Crossref PubMed Scopus (60) Google Scholar, 21Nicholson K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nat. Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (174) Google Scholar, 22Fiebig K.M. Rice L.M. Pollock E. Brunger A.T. Nat. Struct. Biol. 1999; 6: 117-123Crossref PubMed Scopus (237) Google Scholar). The core region of the yeast Q-SNARE complex assembles with a relatively slow rate of ∼6000 m–1 s–1 (21Nicholson K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nat. Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (174) Google Scholar). This rate of Q-SNARE assembly was found to be rate-limiting for the assembly of the ternary complex. Assembly of the synaptic SNAREs is similarly slow. Furthermore, the two synaptic Q-SNAREs syntaxin and SNAP-25 have to interact to allow for subsequent synaptobrevin binding (23Fasshauer D. Antonin W. Subramaniam V. Jahn R. Nat. Struct. Biol. 2002; 9: 144-151Crossref PubMed Scopus (124) Google Scholar). These slow SNARE assembly rates probably allow for a tight control of complex formation in vivo, but to catalyze fast membrane fusion the reaction obviously needs accelerating factors. Strikingly, there are no obvious obstacles such as proline residues or disulfide bonds in the relatively simple four-helix bundle structure that could account for the slow assembly rates. Nevertheless, experiments at elevated temperatures revealed that assembly could not take place in the harsh conditions in which the complex falls apart (23Fasshauer D. Antonin W. Subramaniam V. Jahn R. Nat. Struct. Biol. 2002; 9: 144-151Crossref PubMed Scopus (124) Google Scholar). It seems possible that this marked hysteresis is caused by a rather delicate nucleation reaction, which might render SNARE assembly slow. But what is the structural reason for the delicacy of SNARE nucleation? An answer to this question requires deeper insights into the molecular mechanisms of synaptic SNARE assembly. Therefore, we now have designed fluorescence assays to follow the assembly steps of the core four-helix bundle of the synaptic SNARE complex. Our results suggest that a 1:1 interaction of the Q-SNARE proteins syntaxin and SNAP-25 precedes binding of synaptobrevin. The assembly of the Q-SNAREs is rather complex, since it requires the precise N-terminal nucleation of three helices. Protein Constructs—The basic SNARE expression constructs, cysteine-free SNAP-25A (residues 1–206) (27Fasshauer D. Antonin W. Margittai M. Pabst S. Jahn R. J. Biol. Chem. 1999; 274: 15440-15446Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), the first helix of SNAP-25A (SN1; residues 1–83), the second helix of SNAP-25A (SN2; residues 120–206), the syntaxin 1A SNARE motif (SyxH3; residues 180–262), and synaptobrevin 2 (Syb; residues 1–96) (8Fasshauer D. Eliason W.K. Brünger A.T. Jahn R. Biochemistry. 1998; 37: 10354-10362Crossref PubMed Scopus (208) Google Scholar) have been described before. Shortened versions of these constructs were cloned into the pET28a vector. The following shortened constructs were generated: SNAP-25A, BoNT/A fragment (residues 1–197), SN25ΔC (residues 1–180), ΔNSN25, (residues 39–206), SN1short (residues 7–83), ΔNSN1 (residues 39–83), SN1ΔC (residues 7–66), SN2short (residues 141–204), ΔNSN2 (residues 159–206), and SN1ΔC (residues 141–188); SyxH3, ΔNSyxH3 (residues 212–262) and SyxH3ΔC (residues 183–240); Syb, Syb 1–81 (residues 1–81), SybΔC (residues 1–70), and ΔNSyb (residues 42–96). In addition, we utilized a variety of single cysteine variants of all three SNARE proteins that had been previously characterized by EPR spectroscopy (10Margittai M. Fasshauer D. Pabst S. Jahn R. Langen R. J. Biol. Chem. 2001; 276: 13169-13177Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The following single cysteine variants of SyxH3 (residues 183–262) were used: Cys225, Cys240, and Cys248. Synaptobrevin 2 (residues 1–96) Cys79 and full-length SNAP-25A Cys84 were also used. Protein Purification—The different synaptic SNARE protein fragments were isolated from E. coli and purified by Ni2+-nitrilotriacetic acid affinity chromatography followed by ion exchange chromatography on an Äkta system (Amersham Biosciences) essentially as described (8Fasshauer D. Eliason W.K. Brünger A.T. Jahn R. Biochemistry. 1998; 37: 10354-10362Crossref PubMed Scopus (208) Google Scholar, 27Fasshauer D. Antonin W. Margittai M. Pabst S. Jahn R. J. Biol. Chem. 1999; 274: 15440-15446Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The purity was greater than 95% as determined by SDS-PAGE. All ternary SNARE complexes were assembled overnight from purified individual proteins and purified using a Mono Q-column. Protein concentration was determined by absorption at 280 nm in the presence of guanidine HCl or for the individual SNAP-25 helices and the C-terminally truncated synaptobrevin constructs, which do not posses aromatic residues, using the Bradford assay. Labeling—The sulfhydryl-reactive fluorescent probes anilinonaphthalenesulfonate iodoacetamide (IAANS) Oregon Green 488 iodoacetamide and Texas Red C5 bromoacetamide were purchased from Molecular Probes, Inc. (Eugene, OR). Before labeling, SNARE proteins (∼1–4 mg/ml) containing a single cysteine were dialyzed against degassed PBS buffer (20 mm sodium phosphate, pH 7.4, 100 mm NaCl). About a 10–20-fold molar excess of the respective fluorescence probe in N,N-dimethylformamide was added and incubated for 2 h. The reaction was stopped by adding 10 mm dithiothreitol. After 1 h, excess dye was removed by gel filtration on a Sephadex G-50 column. Afterward, the labeled protein was dialyzed against PBS buffer. The concentration of the labeled protein was determined from its absorption at 327 nm (IAANS), 488 nm (Oregon Green), or 595 nm (Texas Red) using the molar extinction coefficients of the dyes. The protein concentration was determined by the Bradford assay. Fluorescence Spectroscopy—All measurements were carried out in a Fluoromax 2 spectrometer equipped with autopolarizers (Jobin Yvon) in PBS buffer at 25 °C in 1-cm quartz cuvettes. For measurements of IAANS-labeled molecules, the excitation wavelength was 327 nm, and the emission spectrum was measured from 370 to 600 nm. The slit width of the excitation light was set at 2 nm, and that of the emission was set from 2.5 to 4 nm. Initially, the spectrum of the labeled molecule was measured and compared with the spectrum after the addition of unlabeled, interacting protein(s). Kinetic measurements were then performed using the emission wavelength at which the maximum change had been observed. Changes of anisotropy upon complex formation using proteins labeled with Oregon Green were measured at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The slit widths were set to 2–4 nm, and the integration time was set at 1 s. At first, the G factor was determined according to G = IHV/IHH, where I is the fluorescence intensity, and the first subscript letter indicates the direction of the exciting light and second subscript letter is the direction of emitted light. Then the intensities of the vertically and horizontally polarized emission light after excitation by vertically polarized light were measured. The anisotropy (r) was calculated automatically according to r = (IVV – G × IVH)/(IVV + 2 × G × IVH). At the beginning of the reaction, data points were recorded using the smallest interval possible (∼5 s). Later, the intervals were extended accordingly. For fluorescence resonance energy transfer (FRET) measurements, the emission spectrum was recorded from 500 to 700 nm at an excitation of 488 nm. For kinetic measurements, the change of the fluorescence intensity of the acceptor was recorded. CD Spectroscopy—CD measurements were performed using a Jasco model J-720 instrument. For spectral measurements, different protein combinations at a concentration of about 5–10 μm in 20 mm Tris, pH 7.4, 100 mm NaCl (for details, see the appropriate figure legend) were incubated overnight. Hellma quartz cuvettes with a path length of 0.1 cm were used. The spectra were obtained by averaging over 5–50 scans using steps of 0.2 nm with a scan rate of 50 nm/min essentially as described (8Fasshauer D. Eliason W.K. Brünger A.T. Jahn R. Biochemistry. 1998; 37: 10354-10362Crossref PubMed Scopus (208) Google Scholar, 27Fasshauer D. Antonin W. Margittai M. Pabst S. Jahn R. J. Biol. Chem. 1999; 274: 15440-15446Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The measurements were carried out at 25 °C. Kinetic measurements were carried out in PBS buffer using 1-cm quartz cuvettes at 25 °C. Proteins were mixed, and the change in the CD signal was followed at 220 nm. For thermal denaturation experiments, the purified ternary SNARE complexes were dialyzed against PBS buffer. The ellipticity at 220 nm was recorded between 25 and 95 °C at a temperature increment of 30 °C/h. During SNARE assembly, major structural rearrangements occur, with mostly unstructured monomers forming complexes with high α-helical content. This allows us to monitor the assembly reaction using CD spectroscopy (6Fasshauer D. Bruns D. Shen B. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 7: 4582-4590Abstract Full Text Full Text PDF Scopus (169) Google Scholar, 7Fasshauer D. Otto H. Eliason W.K. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 28036-28041Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 23Fasshauer D. Antonin W. Subramaniam V. Jahn R. Nat. Struct. Biol. 2002; 9: 144-151Crossref PubMed Scopus (124) Google Scholar). This approach alone, however, does not provide the spatial information necessary for a detailed analysis of the assembly pathway, since intermediate products already exhibit similar changes in the secondary structure (8Fasshauer D. Eliason W.K. Brünger A.T. Jahn R. Biochemistry. 1998; 37: 10354-10362Crossref PubMed Scopus (208) Google Scholar). To overcome these shortcomings, we have now introduced fluorescence probes into each of the three SNARE complex constituents. Using the crystal structure (11Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1932) Google Scholar) as a guide, several positions in each SNARE protein were chosen for fluorescence labeling. Throughout the study, only the soluble regions of the three synaptic SNAREs that form the core complex were used. An overview of the different labeling positions is given in Fig. 1. A Second Syntaxin Competes with Synaptobrevin for Binding to a 1:1 SyxH3-SNAP-25 Intermediate—In our experiment, we mainly utilized the fluorophore IAANS, which exhibits environmentally sensitive fluorescence properties. Only a few positions of those tested exhibited a pronounced change in the fluorescence intensity that allowed us to follow SNARE assembly. As an example, SNAP-25 labeled at position 84 (SNAP-2584IAANS) showed a strong increase in fluorescence intensity upon binding to syntaxin (Fig. 2B). At pseudo-first-order conditions, the rate of assembly was ∼6000 m–1 s–1 (Fig. 2D). Comparable rates were obtained when the SNARE motif of syntaxin (SyxH3) was labeled with IAANS at position 225, 240, or 248 and mixed with SNAP-25 (∼7600, ∼6800, and ∼7400 m–1 s–1, respectively) (Fig. 2C). Upon titration, saturation of fluorescence was reached at a molar ratio of about two SyxH3 and one SNAP-25 molecule, whereas in the presence of Syb, a molar ratio of 1:1 between SyxH3 and SNAP-25 was observed (data not shown). However, in all experiments in which the reaction between SyxH3 and SNAP-25 was monitored, no clear indication for a two-step assembly process was observable. A possible explanation might be that a transient 1:1 intermediate of SyxH3 and SNAP-25 creates a new binding site with increased affinity for the second SyxH3. In the presence of Syb, the entire reaction appeared to be slower (Fig. 2, A and B) and to consist of at least two phases as we had previously observed using CD spectroscopy (23Fasshauer D. Antonin W. Subramaniam V. Jahn R. Nat. Struct. Biol. 2002; 9: 144-151Crossref PubMed Scopus (124) Google Scholar). As a different read-out for ternary complex formation, we then used Syb labeled at position 79 (Syb79IAANS), which exhibited a clear rise in fluorescence intensity only upon the addition of both SyxH3 and SNAP-25. Interestingly, complex formation was faster when increasing amounts of SNAP-25 were added and SyxH3 was kept at a constant concentration (Fig. 3A), whereas increasing amounts of SyxH3 rather slowed down assembly (Fig. 3B). Thus, it appears that an excess of SyxH3 inhibits Syb binding, most likely because both molecules compete for the same binding site offered by a transient 1:1 SyxH3-SN25 intermediate. Nevertheless, ternary complex formation was only slightly faster when SyxH3 and SNAP-25 were allowed to assemble beforehand (data not shown). This accelerating effect was most obvious when more SNAP-25 than SyxH3 was used. This probably suggests that only a small portion of premixed Q-SNAREs reside as transient 1:1 complexes, whereas the majority appears to exist as stable 2:1 complexes, which do not appear to serve as the primary binding site for synaptobrevin. In another set of experiments, we added SNAP-25 to a mix of SyxH3225OG and SyxH3225TR (i.e. syntaxins labeled at position 225 with the fluorescence dyes Oregon Green or Texas Red, respectively). Although this approach allowed only for a random integration of the differently labeled syntaxins, assembly gave rise to clear FRET, confirming that the Q-SNARE assembly contained two syntaxin molecules (Fig. 4, inset). At pseudo-first-order conditions, a Q-SNARE assembly rate of ∼5900 m–1 s–1 was observed (data not shown), which is similar to the ones assessed with IAANS-labeled Q-SNAREs (see above). Upon the addition of an excess of Syb to the preassembled Q-SNARE complex, the FRET signal disappeared (Fig. 4), suggesting that one of the syntaxin molecules of the preformed 2:1 complex had been exchanged for Syb. The addition of an excess of unlabeled SyxH3 to the preformed Q-SNARE complex led to a comparable disappearance of the FRET signal (Fig. 4), which probably reflects the dissociation of the second syntaxin. Since Syb binding was not faster than the off-rate of the second syntaxin, it is likely that Syb does not actively replace one of the two syntaxins from the 2:1 Q-SNARE assembly. The Syntaxin-SNAP-25 Interaction Nucleates N-terminally—To assess which regions of the SNAREs are sufficient for assembly, N- or C-terminal parts of the SNARE motifs were removed (see Fig. 1). Remarkably, N-terminally truncated SNAP-25 (residues 39–206; ΔNSN25) as well as N-terminally truncated SyxH3 (residues 212–262; ΔNSyxH3) appeared to have lost their ability to undergo Q-SNARE interaction (Fig. 5, A and B). A similar result was obtained by CD spectroscopy, where no increase in α-helical content was observed for a mix of each of the constructs with the respective intact Q-SNARE binding partner (Fig. 5C). Furthermore, these constructs almost completely lost their ability to form a ternary complex containing Syb; only at high concentrations (>5 μm), a stable ternary complex slowly formed (>24 h) (Fig. 5C). This indicates that when the Q-SNARE interaction is perturbed, assembly occurs slowly due to a high energy barrier. In contrast, a C-terminally truncated SyxH3 (residues 183–240; SyxH3ΔC) was still able to interact with SNAP-25 (Fig. 5A). Likewise, a C-terminally truncated SNAP-25 (residues 1–197), which resembles the botulinum neurotoxin A (BoNT/A) cleavage product, formed a complex with SyxH3 (data not shown). However, further shortened SNAP-25 construct (residues 1–180; SN25ΔC, which resembles the BoNT/E cleavage product) only slowly interacted with SyxH3 (Fig. 5B). Nevertheless, upon the addition of Syb, SN25ΔC readily formed a ternary SNARE complex (Fig. 5B). The molecular mass of the purified SyxH3-SN25ΔC complex determined by multiangle laser light scattering indicated a 2:2 stoichiometry (data not shown). An equivalent 2:2 complex has been described for the isolated first helix of SNAP-25 (SN1) and SyxH3 (24Misura K.M. Gonzalez Jr., L.C. May A.P. Scheller R.H. Weis W.I. J. Biol. Chem. 2001; 276: 41301-41309Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Thus, in the absence of Syb, the much shortened second helix of the SN25ΔC construct may not suffice to prevent the competing but slow process of dimerization (for illustration, see Fig. 6). To test whether Syb truncations affected SNARE assembly, we utilized the increase in fluorescence anisotropy of SyxH3225OG upon complex formation. Upon assembly with SNAP-25, anisotropy of SyxH3225OG exhibited only a small increase, which might be caused by the proximity of two OG fluorophores in the SyxH32-SNAP-251 complex. A much more pronounced rise, however, was observed upon ternary SNARE complex formation. Interestingly, N- or C-terminal truncations of Syb had no significant effect on SNARE assembly (Fig. 5D). Similar results were obtained by CD spectroscopy (Fig. 5C). Taken together, these results substantiate the view that Syb binding occurs after Q-SNARE nucleation. Although all of the above SNARE truncations removed a substantial part of the core complex-forming region, about three hydrophobic coiled-coil layers (see Fig. 1), their core complexes remained highly stable (Table I). Consequently, the pronounced effect of the truncations on assembly cannot simply be explained by the fact that the resulting core complex is less stable. Rather, the stability of the Q-SNARE intermediate is likely to be mostly affected. The SNARE complex containing ΔNSyxH3 did not refold during the time period tested, confirming our results that assembly of this fragment is extremely slow (data not shown). Remarkably, complexes containing either N- or C-terminally truncated Syb still exhibited a strong hysteresis (data not shown), suggesting that fragments of the Syb helix are sufficient to produce an "irreversible" SNARE interaction.Table IMelting temperatures of core SNARE complexes containing truncated fragments measured by CD spectroscopyComplexesTemperaturesSx 183-24078 °CSx 212-26272 °CSb 1-7079 °CSb 42-9672 °CSN25ΔC65 °CCore synaptic SNARE complex82 °C (23Fasshauer D. Antonin W. Subramaniam V. Jahn R. Nat. Struct. Biol. 2002; 9: 144-151Crossref PubMed Scopus (124) Google Scholar)SyxH32-SNAP-251 complex44.5 °C at 7 μm (23Fasshauer D. Antonin W. Subramaniam V. Jahn R. Nat. Struct. Biol. 2002; 9: 144-151Crossref PubMed Scopus (124) Google Scholar) Open table in a new tab The Q-SNARE Helices Work Together for Nucleation—The SNARE protein SNAP-25 contains two SNARE motifs connected by a flexible linker region (10Margittai M. Fasshauer D. Pabst S. Jahn R. Langen R. J. Biol. Chem. 2001; 276: 13169-13177Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), which spans the entire length of the SNARE bundle in the core complex. Therefore, the question arises whether both SNAP-25 helices are necessary for successful nucleation of the Q-SNARE complex. For a detailed analysis of this process, we applied the same fluorescence approaches using the two helices of SNAP-25 as independen
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