Syntaxin1a variants lacking an N-peptide or bearing the LE mutation bind to Munc18a in a closed conformation

Abstract

In neurons, soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of synaptic vesicles to the plasma membrane through the formation of a four-helix SNARE complex. Members of the Sec1/Munc18 protein family regulate membrane fusion through interactions with the syntaxin family of SNARE proteins. The neuronal protein Munc18a interacts with a closed conformation of the SNARE protein syntaxin1a (Syx1a) and with an assembled SNARE complex containing Syx1a in an open conformation. The N-peptide of Syx1a (amino acids 1-24) has been implicated in the transition of Munc18a-bound Syx1a to Munc18a-bound SNARE complex, but the underlying mechanism is not understood. Here we report the X-ray crystal structures of Munc18a bound to Syx1a with and without its native N-peptide (Syx1aΔN), along with small-angle X-ray scattering (SAXS) data for Munc18a bound to Syx1a, Syx1aΔN, and Syx1a L165A/E166A (LE), a mutation thought to render Syx1a in a constitutively open conformation. We show that all three complexes adopt the same global structure, in which Munc18a binds a closed conformation of Syx1a. We also identify a possible structural connection between the Syx1a N-peptide and SNARE domain that might be important for the transition of closed-to-open Syx1a in SNARE complex assembly. Although the role of the N-peptide in Munc18a-mediated SNARE complex assembly remains unclear, our results demonstrate that the N-peptide and LE mutation have no effect on the global conformation of the Munc18a-Syx1a complex. membrane trafficking | SM proteins | protein crystallography N eurons communicate across specialized intercellular junctions called synapses. Arrival of an action potential in a presynaptic neuron triggers a calcium (Ca 2+ ) influx, which leads to the exocytosis of presynaptic vesicles and release of neurotransmitters for uptake via receptors in the postsynaptic neuron. Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins drive the fusion of neurotransmitter-containing vesicles to the presynaptic plasma membrane, a critical step in Ca 2+ -triggered exocytosis. Three neuronal SNARE proteinssyntaxin 1a (Syx1a) and synaptosomal-associated protein of 25 kDa (SNAP25), located on the synaptic membrane, and vesicleassociated membrane protein 2 (VAMP2, also called synaptobrevin) combine to form a four-helix bundle, a process that drives membrane fusion. Syx1a contains an N-terminal regulatory region in addition to its C-terminal SNARE domain and adopts two conformations: a closed conformation, in which a three-helix bundle (designated Habc) in the N-terminal region binds intramolecularly to the SNARE domain, and an open conformation in which the SNARE domain binds SNAP25 and VAMP2 to form the ternary SNARE complex.The neuronal Sec1/Munc18a (SM)-like protein Munc18a is essential for synaptic vesicle fusion and associates with the neuronal SNARE proteins in at least two distinct modes. Munc18a

The SM protein Sly1 accelerates assembly of the ER–Golgi SNARE complex

“…However, why do SM proteins use two different binding sites for this purpose? One possibility is that the two binding sites are allosterically coupled to control the accessibility of the bound syntaxin (40,41). It is also possible, although not exclusive, that the two binding sites represent consecutive steps of a reaction cascade or handover mechanism controlled by the SM protein.…”

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

“…2). 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).…”

“…From examination of the crystal structure of Munc18a in complex with Sx1a and its native N-terminus (PDB code: 4jeu), both the N-peptide and the H abc domain of the same Sx1a molecule could be bound to Munc18a at the same time, even in the closed Sx1a conformation (Colbert et al, 2013). Residues 10-26 of Sx1a, which immediately follow the Nfeature articles IUCrJ (2014).…”

“…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.…”