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

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.

Intellectual disability without epilepsy associated with STXBP1 disruption

“…7 Domain-3 together with domain-1 of STXBP1 form the central cavity providing surfaces for Syntaxin-1 binding, an essential step for SNARE complex assembly and subsequent neurotransmitter release. 3,8 No other amino acid altering or splicing mutations were identified in STXBP1 in the remaining patients. We previously sequenced STXBP1 in healthy French Canadian controls (n¼190) and did not identify any amino acid changing or splicing mutations.…”

“…This process is mediated by a complex composed of SNARE proteins and of Munc18-1/STXBP1. [3][4][5] Consistent with its important role for neurotransmission, recent studies showed that mutations in STXBP1 cause severe ID and epilepsy. 6,7 Using array genomic hybridization, Saitsu et al 6 identified a de novo 2 Mb deletion encompassing STXBP1 in a patient with Ohtahara syndrome, an infantile form of epileptic encephalopathy characterized by burst suppressions and severe ID.…”

Reconciling the regulatory role of Munc18 proteins in SNARE-complex assembly

“…Similarly, low-resolution solution scattering models of mouse Munc18a complexed with a soluble C-terminally truncated Sx1a supported a closed binding mode (Colbert et al, 2013). This interaction, which is not compatible with SNARE-complex formation, points to a role for Munc18a as an inhibitor of membrane fusion (Misura et al, 2000;Burkhardt et al, 2008;Chen et al, 2008;Ma et al, 2011). However, other studies have drawn another conclusion: that Munc18 binds open Sx.…”

“…2. Crystal structures of three Munc18:Sx systems have been determined: rat Munc18a:Sx1a (Burkhardt et al 2008), rat Munc18a:Sx1aÁN (Colbert et al, 2013) and a primordial Unc18:Sx1a complex from Monosiga brevicollis (Burkhardt et al, 2011). These crystal structures show the same closed Sxbinding mode when a soluble C-terminally truncated form of Sx1a was used to generate crystals.…”

“…Structural studies on neuronal SM proteins have suggested that Munc18a plays a negative regulatory role in membrane fusion by binding to closed Sx1a, thereby preventing SNAREcomplex formation (Burkhardt et al, 2008(Burkhardt et al, , 2011Colbert et al, 2013). However, deletion (Verhage et al, 2000) or knockdown ) of Munc18a in neurons completely abolishes secretory vesicle fusion and neurotransmitter release, suggesting it plays a positive regulatory role.…”