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Abstract
The membrane fusion necessary for vesicle trafficking is driven by the assembly of heterologous SNARE proteins orchestrated by the binding of Sec1/Munc18 (SM) proteins to specific syntaxin SNARE proteins. However, the precise mode of interaction between SM proteins and SNAREs is debated, as contrasting binding modes have been found for different members of the SM protein family, including the three vertebrate Munc18 isoforms. While different binding modes could be necessary, given their roles in different secretory processes in different tissues, the structural similarity of the three isoforms makes this divergence perplexing. Although the neuronal isoform Munc18a is well-established to bind tightly to both the closed conformation and the N-peptide of syntaxin 1a, thereby inhibiting SNARE complex formation, Munc18b and -c, which have a more widespread distribution, are reported to mainly interact with the N-peptide of their partnering syntaxins and are thought to instead promote SNARE complex formation. We have reinvestigated the interaction between Munc18c and syntaxin 4 (Syx4). Using isothermal titration calorimetry, we found that Munc18c, like Munc18a, binds to both the closed conformation and the N-peptide of Syx4. Furthermore, using a novel kinetic approach, we found that Munc18c, like Munc18a, slows down SNARE complex formation through high-affinity binding to syntaxin. This strongly suggests that secretory Munc18s in general control the accessibility of the bound syntaxin, probably preparing it for SNARE complex assembly.
Highlights
The membrane fusion necessary for vesicle trafficking is driven by the assembly of heterologous SNARE proteins orchestrated by the binding of Sec1/Munc18 (SM) proteins to specific syntaxin SNARE proteins
Using a novel kinetic approach, we found that Munc18c, like Munc18a, slows down SNARE complex formation through high-affinity binding to syntaxin
The sequence conservation pattern reveals that both spatially separated binding regions for syntaxins, the N-peptide binding pocket and the central cavity, which binds to the closed conformation, are maintained in all Munc18s, suggesting that the binding mode that includes two binding surfaces is preserved in the three vertebrate isoforms
Summary
We extended our previous analysis on the phylogeny of Munc18s from all animals and their single-celled ancestors [11]. These results suggest that two regions of Syx, the N-peptide and the remainder in a closed conformation, bind to Munc18c This binding mode resembles that of Munc18a and Syx1a, but it is unclear whether Munc18c exerts a control similar to that of Munc18a over the ability of the bound syntaxin to engage in SNARE complex formation. When Munc18c was added to the SNARE assembly reaction, we observed a clear inhibition of ternary SNARE complex formation (Fig. 3), the inhibition exerted by Munc18c appeared to be somewhat less pronounced than the inhibition of neuronal SNARE complex formation by Munc18a reported earlier [10] and can probably be attributed to a faster dissociation of the Munc18c-Syx complex compared with the Munc18a-Syx1a complex (supplemental Fig. S4) This finding corroborates the idea that Munc18c, very like Munc18a, binds to the closed conformation of its syntaxin partner and thereby inhibits its ability to engage in SNARE complex interactions. The mutations in the linker helix of syntaxins do not bypass the tight controlling effect of Munc18c, hinting at a subtle structural and functional difference between Munc18a and Munc18c
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