Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed-open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1-N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.membrane trafficking | protein-peptide interaction | protein-protein interaction | Sec/Munc protein S ec/Munc (SM) and soluble NSF attachment protein receptor (SNARE) proteins play fundamental roles in regulating membrane traffic (1-3). The cognate interacting partners comprising the SM protein Munc18-1 and the SNARE protein Syntaxin1 (Sx1) are of special importance to human physiology because they regulate synaptic vesicle-mediated neurotransmitter release (4). Two alternate binding modes have been described for this pair of proteins. One mode involves Munc18-1 interacting with "closed" Sx1, in which the SNARE H3 helical motif is sequestered by the three Habc helices of Sx1 to form a four-helix bundle (5, 6) ( Fig. 1). This closed binding mode is consistent with a negative regulatory role for Munc18-1 because the SNARE H3 helix in closed Sx1 is unable to interact with SNARE partners to form the complexes that drive vesicle fusion (7) (Fig. 1A). However, the closed binding mode of Syntaxin is not universal and may be a specialization of regulated exocytosis (8). A second binding mode, which likely underpins a general function of SM proteins, occurs when Sx1 is in an "open" conformation (i.e., when the H3 helix is separated from the Habc helices) in the SNARE ternary complex (9, 10) (Fig. 1A). This binding mode is dependent on the very N-terminal 10 residues of Sx1, the N peptide. This second mode is consistent with a positive regulatory role for Munc18-1, because SNARE ternary complex formation is required for vesicle fusion. The N-peptide interaction has been characterized structurally for the highly homologous protein pair of Munc18-3 and Syntaxin4 (Sx4) (11), which regulate trafficking of the insulin-stimulated glucose transporter GLUT4 in muscle and fat cells (12).Munc18 proteins contribute to s...

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...

SummaryMunc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a 'closed', fusion-incompetent to an 'open', fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1 D317-333) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1 D317-333to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1 D317-333 binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1 D317-333 could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.

The cholesterol-dependent cytolysins (CDCs) are a family of bacterial toxins that are important virulence factors for a number of pathogenic Gram-positive bacterial species. CDCs are secreted as soluble, stable monomeric proteins that bind specifically to cholesterol-rich cell membranes, where they assemble into well-defined ring-shaped complexes of around 40 monomers. The complex then undergoes a concerted structural change, driving a large pore through the membrane, potentially lysing the target cell. Understanding the details of this process as the protein transitions from a discrete monomer to a complex, membrane-spanning protein machine is an ongoing challenge. While many of the details have been revealed, there are still questions that remain unanswered. In this review, we present an overview of some of the key features of the structure and function of the CDCs, including the structure of the secreted monomers, the process of interaction with target membranes, and the transition from bound monomers to complete pores. Future directions in CDC research and the potential of CDCs as research tools will also be discussed.

The cholesterol-dependent cytolysin (CDC) genes are present in bacterial species that span terrestrial, vertebrate, and invertebrate niches, which suggests that they have evolved to function under widely different environmental conditions. Using a combination of biophysical and crystallographic approaches, we reveal that the relative stability of an intramolecular interface in the archetype CDC perfringolysin O (PFO) plays a central role in regulating its pore-forming properties. The disruption of this interface allows the formation of the membrane spanning β-barrel pore in all CDCs. We show here that the relative strength of the stabilizing forces at this interface directly impacts the energy barrier posed by the transition state for pore formation, as reflected in the Arrhenius activation energy (Ea) for pore formation. This change directly impacts the kinetics and temperature dependence of pore formation. We further show that the interface structure in a CDC from a terrestrial species enables it to function efficiently across a wide range of temperatures by minimizing changes in the strength of the transition state barrier to pore formation. These studies establish a paradigm that CDCs, and possibly other β-barrel pore-forming proteins/toxins, can evolve significantly different pore-forming properties by altering the stability of this transitional interface, which impacts the kinetic parameters and temperature dependence of pore formation. IMPORTANCE The cholesterol-dependent cytolysins (CDCs) are the archetype for the superfamily of oligomeric pore-forming proteins that includes the membrane attack complex/perforin (MACPF) family of immune defense proteins and the stonefish venom toxins (SNTX). The CDC/MACPF/SNTX family exhibits a common protein fold, which forms a membrane-spanning β-barrel pore. We show that changing the relative stability of an extensive intramolecular interface within this fold, which is necessarily disrupted to form the large β-barrel pore, dramatically alters the kinetic and temperature-dependent properties of CDC pore formation. These studies show that the CDCs and other members of the CDC/MACPF/SNTX superfamily have the capacity to significantly alter their pore-forming properties to function under widely different environmental conditions encountered by these species.