The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion

Plasmid construction and site-directed mutagenesis

Full-length syntaxin 1A (amino acids 1–288) was inserted into pET-28b vector to make N-terminal His₆-tagged protein. Full-length and soluble VAMP2 (amino acids 1–116 and 1–94) were inserted into pGEX-KG vector to make N-terminal glutathione S-transferase (GST) fusion proteins. Recombinant syntaxin 1A without the Habc domain (STX1A HT, amino acids 168–288) and wild-type SNAP-25, truncated SNAP-25A (Δ9) and SNAP-25E (Δ26) (amino acids 1–206, 1–197 and 1–180, respectively) were also made as GST fusion proteins. To introduce unique cysteine at the specific position of SNAP-25 and syntaxin 1A, the native cysteines C85, 88, 90 and 92 in SNAP-25 and C145, 271 and 272 in syntaxin 1A were changed to alanines. All cysteine mutants were generated by QuikChange site-directed mutagenesis kit (Stratagene, Cedar Creek, Texas, USA), and were confirmed by sequencing.

Protein expression, purification, and spin labeling

GST fusion proteins were expressed in E. coli Rosetta (DE3) pLysS (Novagene, Darmstadt, Germany) and purified by glutathione-agarose beads (Sigma, Munich, Germany). The cells were grown at 37 °C in LB with glucose (2 g/liter), ampicillin (100 µg/ml), and chloramphenicol (50 µg/ml) until A₆₀₀ reached 0.6–0.8. After adding 0.3 mM isopropylthio-β-D-galactopyranoside (IPTG), the cells were further grown for 6 h at 22 °C for VAMP2 and SNAP-25 but at 16 °C for syntaxin 1A. GST fusion protein purification was performed following the procedure described in elsewhere (Lu, Song & Shin, 2010). We add 1% n-octyl-glucoside (OG) in the cleavage buffer for syntaxin 1A and VAMP2.

His₆-tagged syntaxin 1A were expressed in E. coli BL21(DE3) CodonPlus-RIL (Stratagene) and purified using Ni-NTA resin (Qiagen, Hilden, Germany). The cells were grown at 37 °C in LB with glucose (2 g/liter), kanamycin (34 µg/ml), and chloramphenicol (50 µg/ml) until A₆₀₀ reached 0.6–0.8. Protein expression was induced by 0.5 mM IPTG, and the cells were grown for an additional 4–6 h at 16 °C. His₆-tagged protein purification was performed following the procedure described in elsewhere (Lu et al., 2014). Finally, the protein was eluted from the Ni-NTA resin by elution buffer (25 mM HEPES, 100 mM KCl with 250 mM imidazole, pH 7.4).

The cysteine mutants of SNAP-25 and syntaxin 1A were reacted with (1-oxyl-2,2,5,5-tetramethylpyrrolinyl-3-methyl) methanethiosulfonate (MTSSL) spin label at 4 °C overnight while the protein was bound to the GST-agarose beads. To remove free spin label, the beads with bound proteins were extensively washed with the cleavage buffer then cleaved by thrombin (Sigma, Munich, Germany). The spin-labeling efficiency was determined by the 50 µM 2,2,6,6-tetramethyl-4-piperidine N-oxide (TEMPO) standard. For all samples, the efficiency was ∼80%.

Pull-down method preparing for the binary and ternary complex

Details of SNARE complex formation by pull-down method have been described (Lu, Song & Shin, 2010). Briefly, purified His₆-tagged syntaxin 1A was first added to the Ni-NTA resin solution and incubated for 1 h at room temperature. After washing out the free proteins, 2-fold excess of purified GST-SNAP-25 or 2-fold excess of GST-SNAP-25 and 4-fold excess of soluble VAMP2 were mixed with His₆-syntaxin 1A to form the t-SNAREs binary complex or trans-SNARE ternary complex, respectively. The mixture was incubated at 4 °C overnight. After extensive washing to remove the unbound proteins, the complex was eluted with a buffer containing 250 mM imidazole and 1% n-octyl-glucoside (OG). The formation of SNARE complex was confirmed with SDS-PAGE gel.

Vesicle preparation and membrane reconstitution

Large unilamellar vesicles (LUVs) of 100 nm diameter were prepared using an extruder as described previously (Lu, Song & Shin, 2010). The lipid-to-protein molar ratio was 200:1 (except as noted). For the lipid mixing assay, the mixture of POPC and DOPS in a molar ratio of 85:15 was used. Additionally, NBD-PS and rhodamine-PE (1.5 mol% each) were added to the v-SNARE vesicles for fluorescence detection of lipid mixing. The t-SNAREs were preformed by mixing syntaxin1A HT and SNAP-25 in a molar ratio of 1:1 at room temperature for 60 min before reconstitution. In all cases, proteins were reconstituted by using the dialysis method as described previously (Lu et al., 2014).

Fluorescence lipid mixing assay

The v-SNARE vesicles were mixed with the t-SNARE vesicles in a molar ratio of 1:9 for the total lipid mixing assay. The final solution for each reaction contained about 1 mM lipids in HEPES buffer (25 mM HEPES, 100 mM KCl, pH 7.4) with a total volume of 100 µl. Fluorescence intensity was measured at the NBD’s excitation and emission wavelengths of 465 and 530 nm, respectively. Fluorescence signals were recorded by a Varian Cary Eclipse model fluorescence spectrophotometer using a quartz cell of 100 µl with 2 mm path length. After 3,600 s, 0.1% (vol/vol) reduced Trion X-100 (Sigma, Munich, Germany) was added to obtain the maximum fluorescence intensity (MFI). All measurements were performed at 35 °C.

EPR data collection

EPR spectra were obtained using a Bruker ESP 300 spectrometer (Bruker, Bremen, Germany) equipped with a low noise microwave amplifier (Miteq, Hauppauge, New York, USA) and a loop-gap resonator (Medical Advances, Milwaukee, Wisconsin, USA). The modulation amplitude was set to be no greater than one-fourth of the line width. The spectra were collected at room temperature in the first-derivative mode with 1 mW microwave power. The detailed EPR samples preparation followed the methods as described (Lu et al., 2014; Lu, Song & Shin, 2010).

The conformation of the C-terminus of SNAP-25 SN1 and SN2

To compare the SN1 and SN2 conformations in the t-SNAREs binary complex, we employed site-directed spin labeling (SDSL) and continuous wave (CW) EPR spectroscopy and prepared three cysteine mutants located in the C-terminus of SN1 and SN2, respectively (Fig. 1A): C63, 70 and 77 of SNAP-25 SN1, and C184, 191 and 198 of SNAP-25 SN2. The selected residues were all at the predicted “g” position in the heptad repeats of SNAP-25 SNARE motifs (Poirier et al., 1998). In order to avoid the oligomerization of syntaxin 1A, we used His₆-tagged full-length syntaxin 1A to pull down the spin-labeled GST-tag SNAP-25. Two-fold excess of purified SNAP-25 were incubated with syntaxin 1A, and then washed extensively to get rid of the free SNAP-25.

The EPR spectra (Fig. 1B) clearly showed that each position in the C-terminal of SN1 had a broad lineshape, especially when the complex reconstituted in the membrane. That indicated that these positions were all interacting with syntaxin 1A, forming the ordered binary complex. However, at the corresponding positions located in the C-terminal of SN2, the EPR lineshape became much sharper, which indicated that these positions may not be completely involved in the binary complex. Based on these data, we propose that SN1 of SNAP-25 and syntaxin 1A form a highly ordered structure, while the C-terminal of SN2 is unstructured. This kind of binary complex is much closer to the active t-SNAREs binary complex in vivo, which has 1:1 composition of syntaxin 1A and SNAP-25 (An & Almers, 2004).

We further investigated the C-terminal conformation of SNAP-25 SN2 in the ternary SNARE complex. His₆-tagged syntaxin 1A was incubated with two-fold excess of spin-labeled GST-tag SNAP-25 and four-fold excess of GST-tag soluble VAMP2. At three C-terminal positions (C177, 184 and 198), when adding soluble VAMP2, all the EPR spectra became broader, indicating that the C-terminal of SNAP-25 SN2 interacted with VAMP2 and formed the trans-SNARE ternary complex (Fig. 2). However, we still detected some sharp components at these positions, which may be because the trans-SNARE complex easily splays out at the end of the C-terminus. This also implies that the complete SNARE assembly may require other regulatory proteins (such as synaptotagmin and complexin) at the late zippering step to promote the tight SNARE bundle formation, which is crucial for fusion pore opening.

The C-terminus of SNAP-25 is essential for synaptic membrane fusion

The EPR lineshape analysis revealed that the C-terminal of SNAP-25 SN2 in the binary complex is partially free, and that the sharp components can be detected even in the ternary complex. One might then wonder whether the formation of the completely assembled SNARE complex is required for membrane fusion. To address this question, we examined the fusion activity of the nitroxide spin-labeled SNAP-25 using the fluorescence lipid mixing assay. The SNAREs reconstituted vesicles that were employed in the study are shown in Fig. 3. The nitroxide side chain was relatively bulky, similar in size to that of tryptophan. Therefore, if the formation of the completed coiled coil is necessary for membrane fusion, the alterations at the internal positions (‘a’ or ‘d’) (Poirier et al., 1998) might cause some serious perturbations. As Fig. 3 shows, we detected the distinct lipid mixing activity decreasing with the introduction of the mutations, as compared to the wild-type SNAP-25. Moreover, when the spin-labeled position was closer to the central of SNAP-25 SNARE motif, the perturbation was stronger. SNAP-25 C188 showed a 78% fusion activity of wild-type SNAP-25, while SNAP-25 C181 was just left of 40%. Therefore, our data confirm that SN2 of SNAP-25 is required for activating membrane fusion.

The different SDS-resistant abilities of the truncated SNARE complex

Former studies show that SNARE proteins can form a very stable complex that is resistant to SDS denaturation and high temperatures (>90 °C) (Hayashi et al., 1995; Wei et al., 2000). Therefore, we compared the SDS resistant property of different SNARE complexes, composed of truncated SNAP-25, syntaxin 1A and VAMP2 (Fig. 4). We mixed syntaxin 1A and wild-type SNAP-25, or truncated SNAP-25A (Δ9) or SNAP-25E (Δ26) at the molar ratio of 1:2, and with four different amounts of soluble VAMP2 (1-, 2-, 4-, 6-fold of syntaxin 1A) at room temperature for one hour, and ran the SDS-PAGE gel without boiling. As expected, the wild-type SNAP-25 formed the SDS-resistant complex with the other two SNARE proteins (Fig. 4A, red arrow). We also found that there were many high molecular weight complexes besides the monomeric SNARE complex, which may be due to the aggregation of SNARE complexes by SNAP-25 swapping (Fig. 4A, red bracket). SNAP-25A (Δ9) formed a SDS-resistant mono-complex and small amounts of high molecular weight complexes (Fig. 4B), while SNAP-25E (Δ26) did not form any SDS-resistant complex (Fig. 4C). Therefore, these results suggest that the SNARE complexes composed of the truncated SNAP-25 display a low stability to the SDS detergent.

The fusion activity of the truncated SNAP-25 mutants

The less stable truncated SNARE complexes, due to the truncated SNAP-25, may decrease their fusion activity. To test this idea, we used the fluorescence lipid mixing assay to detect the function of the truncated SNAP-25. As Figs. 5A and 5B show, the truncated SNAP-25E (Δ26) formed SNARE complex totally abolished the function to induce membrane fusion, just as in the case of negative control, which has no SNAP-25. The SNAP-25A (Δ9) mutant had only about 6% of maximum fluorescence intensity (MFI), while the wild-type SNAP-25 had about 20% of MFI. So, it is clear that the truncated SNAP-25A lost most of its function, although it could still form the SDS-resistant SNARE complex (Fig. 4B). Our lipid mixing results were also in agreement with the in vivo studies, which showed the same tendency of different membrane fusion activities in neurotoxins treated spinal cord cell cultures (Bergey, Bigalke & Nelson, 1987; Williamson, Fitzgerald & Neale, 1992).

The local structure of the truncated SNARE complex

We are very interested in exploring why there was such a big difference between the wild-type and truncated SNARE complexes. Therefore, we employed CW-EPR spectroscopy to investigate the local conformational changes of the truncated ternary SNARE complex. The spin-labeled syntaxin 1A proteins were incubated with soluble VAMP2 and the truncated SNAP-25 mutants, and then formed ternary complexes. We selected four positions in syntaxin 1A, corresponding to the upstream positions of SNAP-25 neurotoxins cleavage sites (N-terminus). As Figs. 6A and 6B show, in the N-terminal SNARE motif of syntaxin 1A, the spectra were all very broad, both for the wild-type and the truncated SNARE complexes. This suggests that SNARE proteins form the complete coiled coil regardless of the kind of truncated SNAP-25 involved. Since the BoNT/A and BoNT/E cleavage sites were both at a distance from these spin-labeled positions, the truncated SNAP-25 did not disrupt the SNARE core formation.

However, when the syntaxin 1A spin-labeled positions were changed to the last four positions of its SNARE motif (C-terminus), the EPR spectra became much sharper (Figs. 6C and 6D), which reflected the fast motion of nitroxide and indicated that this region is largely unstructured. So, our structural data show that the truncated SNAP-25 mutants induced the partially free conformation of the SNARE core complex in the C-terminus. This tendency was much more obvious when the ternary complex was reconstituted in the membrane, indicating the function of the membrane in the arrangement of the SNARE complex.