Low-resolution solution structures of Munc18:Syntaxin protein complexes indicate an open binding mode driven by the Syntaxin N-peptide

Abstract

When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Munc18 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxin1a interacts with Munc18-1 whether or not the Syntaxin1a N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxin1a and Syntaxin4 adopt extended conformations in complexes with their respective Munc18 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in Munc18:Syntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Munc18-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode. membrane fusion | protein interactions | small-angle neutron scattering | small-angle X-ray scattering M embrane trafficking is an essential and highly regulated process whereby cargo-carrying vesicles are directed to dock and fuse with specific cell membranes. The process requires soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins on vesicle and target membranes. These interact to form high-affinity SNARE complexes required for docking and membrane fusion (1, 2). Structurally, the SNARE complex is a parallel four-helix bundle comprising helices contributed from several partner SNAREs (3). This helical bundle promotes vesicle fusion by bringing the vesicle and plasma membranes into close proximity and by providing the energy required for membrane fusion (4, 5).In the neuronal synapse, neurotransmitters are released from nerve cells by SNARE-mediated fusion of vesicles with the plasma membrane in response to action potentials. In this process, one of four SNARE helices is contributed by Syntaxin (Sx) 1a, a protein localized at the plasma membrane by a transmembrane helix. In addition to its membrane-anchored SNARE helix (H3), Sx1a has an Habc domain that can bind to the H3 helix (6) forming a closed Sx1a conformation (Fig. 1A). This closed conformation inactivates Sx1a by preventing H3 inter...

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

“…This explanation accords with present models for SM interactions with several Qa-SNAREs (Rathore et al, 2010;Rizo and Südhof, 2012;Archbold et al, 2014). It is consistent, too, with recent studies of the yeast and mammalian SM proteins Sly1p (Demircioglu et al, 2014) and Munc18-1 (Christie et al, 2012), which appear to bind their cognate Qa-SNAREs concurrently via the major cleft and the N-terminal site on the SM protein. In the case of Sly1p, binding to the N-terminal site has been suggested to be important for forming a quasi-open or "loosened" conformation of the Qa-SNARE.…”

Comparative studies of Munc18c and Munc18-1 reveal conserved and divergent mechanisms of Sec1/Munc18 proteins

“…Our data demonstrate that unlike Munc18-1, Munc18c binding to syntaxin does not block SNARE complex assembly or the fusion kinetics. When bound to Munc18c, syntaxin-4 adopts an open conformation fully competent for assembly with other SNARE subunits, in agreement with Munc18c structural and binding studies performed in solution (57,58). Therefore, the closed syntaxin binding mode is not conserved even among exocytic SM proteins.…”

“…The structures of SNARE complexes are conserved across vesicle fusion pathways (72)(73)(74). Similarly, all SM proteins adopt a compact, arch-like configuration (34,35,58,75,76). However, the architecture of the SNARE/ SM complex appears to differ significantly across vesicle fusion pathways.…”

“…It has been proposed that the Munc18c/syntaxin-4 dimer precludes the assembly of the t-SNARE complex (41). However, solution structural data suggest that Munc18c-bound syntaxin-4 does not adopt a closed conformation (58). Here, we sought to determine whether Munc18c negatively regulates SNARE-mediated fusion using full-length proteins in a membrane environment.…”

Crucial role of the hydrophobic pocket region of Munc18 protein in mast cell degranulation

“…Indeed, a recent study has demonstrated that the crucial role of syntaxin N-peptide in monomeric interaction with Munc18 is isoform-dependent; deletion of N-peptide in syntaxin-1 has little effect on its binding to Munc18-1, whereas a similar deletion in syntaxin-4 dramatically reduces its binding with Munc18-3 (46). Mechanistically, we suggest that these two binding modes work together in chaperoning the cognate syntaxins in both neuronal and immune cell exocytosis; however, the relative contributions of these two binding modes vary depending on cell types.…”