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.2008 Apr 9;27(7):923-33.
doi: 10.1038/emboj.2008.37. Epub 2008 Mar 13.

Munc18a controls SNARE assembly through its interaction with the syntaxin N-peptide

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Munc18a controls SNARE assembly through its interaction with the syntaxin N-peptide

Pawel Burkhardt et al. EMBO J..

Abstract

Sec1/Munc18-like (SM) proteins functionally interact with SNARE proteins in vesicular fusion. Despite their high sequence conservation, structurally disparate binding modes for SM proteins with syntaxins have been observed. Several SM proteins appear to bind only to a short peptide present at the N terminus of syntaxin, designated the N-peptide, while Munc18a binds to a 'closed' conformation formed by the remaining portion of syntaxin 1a. Here, we show that the syntaxin 16 N-peptide binds to the SM protein Vps45, but the remainder of syntaxin 16 strongly enhances the affinity of the interaction. Likewise, the N-peptide of syntaxin 1a serves as a second binding site in the Munc18a/syntaxin 1a complex. When the syntaxin 1a N-peptide is bound to Munc18a, SNARE complex formation is blocked. Removal of the N-peptide enables binding of syntaxin 1a to its partner SNARE SNAP-25, while still bound to Munc18a. This suggests that Munc18a controls the accessibility of syntaxin 1a to its partners, a role that might be common to all SM proteins.

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Figures

Figure 1
Figure 1
The N-peptide participates in binding of syntaxin 1a to Munc18a. Calorimetric titrations of Syx1a (1–262 or 25–262) into Munc18a. Shown are the integrated areas normalized to the amount of syntaxin 1a (kcal/mol) versus the molar ratio of syntaxin to Munc18a. The solid lines represent the best fit to the data for a single binding site model using a nonlinear least squares fit.
Figure 2
Figure 2
Newly refined crystal structure of the Munc18a–syntaxin1a complex. (A) Munc18a domains 1, 2, and 3 are defined as described (Misuraet al, 2000) and coloured blue, green, and yellow, respectively. The H3 SNARE domain of syntaxin is coloured purple, and the regulatory Habc domain and N-terminal peptide are coloured red. The dashed line represents residues 10–26 of syntaxin, which are not visible in the electron density maps. (B)Fo–Fc omit map. Positive electron density is shown in green at a 3.0 σ contour. The syntaxin N-terminal peptide is shown in stick representation in dark salmon, and a symmetry-related peptide in the crystal is shown in light grey. The map was calculated by omitting the peptide during final round of refinement.
Figure 3
Figure 3
Comparison of syntaxin N-terminal peptide-binding sites on (A) Munc18a and (B) Munc18c. In each case, the Munc18 homologue is shown in surface representation, with the surface formed by atoms that contact syntaxin coloured orange. Residues that form hydrogen bonds with syntaxin are shown beneath the surface, with the hydrogen bonds shown as dashed lines. In (A), the syntaxin 1A peptide is coloured dark salmon. In (B), the syntaxin 4 is light blue. Residues 10–19, which were only observed in the Munc18c-syntaxin4 structure, are not shown. Note that in (B), Glu233, which is located in the α8 loop and interacts with Asp3, is not shown for clarity.
Figure 4
Figure 4
Removal of the N-peptide of syntaxin allows for SNARE complex formation of Munc18-bound syntaxin. (A, B) Assembly of SNARE complexes in the absence or presence of Munc18a was monitored by the formation of SDS-resistant complexes containing synaptobrevin (Syb1–96) labelled with the fluorescent dye Alexa-488 at Cys79. For both syntaxin 1a variants, Syx1a (1–262) and Syx1a (25–262), SNARE complexes formed in the absence of Munc18a. In the presence of Munc18a, however, SNARE complex formation was abolished for Syx1a (1–262) (A), whereas a clear SDS-resistant band was visible for Syx1a (25–262) (B). Note that the SDS-resistant band in the presence of Munc18a appears to be weaker than that in the absence of Munc18a. This might be due to the fact Munc18a, which runs at the same molecular mass as the SDS-resistant SNARE complex, interfered with the intensity of the fluorescent band. (C–E) Ternary SNARE complex formation was followed by the increase in fluorescence anisotropy of 40 nM fluorescent Syb1-96 upon mixing with 500 nM syntaxin 1a and 750 nM SNAP-25. Munc18a (750 nM) inhibited SNARE complex formation for Syx1a (1–262) (B), but not for Syx1a (25–262) (D) and the ‘open' syntaxin variant SyxLE (E). Note that for Syx1a (25–262) SNARE complex assembly occurred at about similar speed as in the absence of Munc18a.
Figure 5
Figure 5
The two binding sites Vps45 and syntaxin 16. Calorimetric titrations of syntaxin 16 (1–302 or 1–27) into Vps45. Shown are the integrated areas normalized to the amount of syntaxin 16 (kcal/mol) versus its molar ratio to Vps45. The solid lines represent the best fit to the data for a single-binding-site model using a nonlinear least squares fit.
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