Munc18-1 induces conformational changes of syntaxin-1 in multiple intermediates for SNARE assembly

Abstract

In neuronal exocytosis, SNARE assembly into a stable four-helix bundle drives membrane fusion. Previous studies have revealed that the SM protein Munc18-1 plays a critical role for precise SNARE assembly with the help of Munc13-1, but the underlying mechanism remains unclear. Here, we used single-molecule FRET assays with a nanodisc membrane reconstitution system to investigate the conformational dynamics of SNARE/Munc18-1 complexes in multiple intermediate steps towards the SNARE complex. We found that single Munc18-1 proteins induce the closed conformation of syntaxin-1 not only in the free syntaxin-1 but also in the t-SNARE (syntaxin-1/SNAP-25) complex. These results implicate that Munc18-1 may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc18-1. Furthermore, the kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/ Munc18-1 complex is less stable than that in the closed syntaxin-1/Munc18-1 complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex. Neuronal exocytosis for neurotransmitter release is driven by the assembly of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are syntaxin-1 and SNAP-25 in the presynaptic plasma membrane and synaptobrevin in the synaptic vesicle membrane 1,2. The assembly of these proteins generates a stable four-helix bundle between the vesicle and target membranes, thus promoting membrane fusion 3,4. Although the membrane fusion can be induced by the SNAREs alone in vitro, a number of auxiliary proteins are required for membrane fusion with high speed and high fidelity in vivo 5,6. Among those, the Sec1/Munc18 (SM) family proteins Munc18-1 and Munc13-1 are known to be essential for SNARE-mediated membrane fusion 7-9. Extensive studies on roles of the Munc18-1 and Munc13-1 in the membrane fusion have established that these proteins are critically involved in the regulation of SNARE complex formation. Initially, Munc18-1 induces the closed conformation of syntaxin-1, locking the syntaxin-1 protein in a cleft of Munc18-1, that inhibits the spontaneous binding of SNAP-25 to syntaxin-1 10,11. Recent reports have revealed that the MUN domain of Munc13-1 promotes the transition from the syntaxin-1/Munc18-1 complex to the ternary SNARE complex in the presence of SNAP-25 and synaptobrevin, suggesting that Munc13-1 plays a role in opening syntaxin-1 for the subsequent SNARE assembly 12-15. On the other hand, it is also known that Munc18-1 stimulates membrane fusion when it binds to a fully assembled SNARE complex 16-18. Despite such major advances, many important questions concerning the mechanisms underlying the precise regulation of SNARE complex formation by the Munc18-1 and Munc13-1 still remain unanswered. For instance,

Conformational Change of Syntaxin-3b in Regulating SNARE Complex Assembly in the Ribbon Synapses

“…Therefore, during SNARE complex assembly, the syntaxin/Munc18 complex requires a brain-specific priming factor, such as Munc13-1, to catalyze the transition of the Habc domain of syntaxin to an open conformation, exposing the C-terminal SNARE motif and allowing for the initiation of SNARE complex formation in the presence of SNAP-25 and synaptobrevin 15, 16, 18, 20 . Recent single-molecule fluorescence studies have demonstrated the conformation of isolated syntaxin-1a in a dynamic transition between an open and closed conformation, despite the complete closed conformation seen by NMR and SAXS data 15, 17, 21, 24, 40, 41 . Despite the importance of the conformational switch of syntaxin in controlling synaptic vesicle exocytosis, the underlying molecular mechanism of other isoforms has not been investigated.…”

Relative enrichment – a density-based colocalization measure for single-molecule localization microscopy

“…In contrast, Munc18-1 exclusively orchestrate the assembly of release machinery [21]. Evidence from previous studies in neuroendocrine cells suggest the two proteins colocalize at docked vesicles and disperse following vesicular neurotransmitter release [22][23][24][25].…”

Role of Munc18-1 in the biological functions and pathogenesis of neurological disorders (Review)

“…The N-terminal peptide is essential for vesicle fusion itself, whilst the Habc domain regulates this fusion by forming a closed syntaxin-1 conformation ( 20 , 21 ). The current hypothesis is that Munc18-1 and syntaxin-1 have several binding modes: i) Munc18-1 binds to syntaxin-1 (via the Habc domain) in a ‘closed’ conformation, stabilizing the conformation of syntaxin-1 and inhibiting the assembly of the SNARE complex ( 16 ); ii) Munc18-1 combined with ‘closed’ conformation syntaxin-1 serves as a template for SNARE assembly, with Munc13-1 helping to open the syntaxin-1 ‘closed’ conformation. Munc13-1 Bridges synaptic vesicles and presynaptic membrane fusion, and Munc18-1 and Munc13-1 coordinate the assembly of the T-SNARE complex together in an NSF-SNAP resistant manner ( 22 ); and iii) Munc18-1 combined with the ‘open’ conformation ofsyntaxin-1 (N-terminal short peptide) initiates and stimulates SNARE-mediated membrane fusion ( 16 ).…”

“…The current hypothesis is that Munc18-1 and syntaxin-1 have several binding modes: i) Munc18-1 binds to syntaxin-1 (via the Habc domain) in a ̔closed̓ conformation, stabilizing the conformation of syntaxin-1 and inhibiting the assembly of the Snare complex (16); ii) Munc18-1 combined with ̔closed̓ conformation syntaxin-1 serves as a template for Snare assembly, with Munc13-1 helping to open the syntaxin-1 ̔closed̓ conformation. Munc13-1 Bridges synaptic vesicles and presynaptic membrane fusion, and Munc18-1 and Munc13-1 coordinate the assembly of the T-Snare complex together in an nSF-SnaP resistant manner (22); and iii) Munc18-1 combined with the ̔open̓ conformation ofsyntaxin-1 (n-terminal short peptide) initiates and stimulates Snare-mediated membrane fusion (16). The regulatory mechanism of Munc18-1 is complex, involving promotion and inhibition of Snare assembly; however, the promotion process is the more dominant of the two.…”

“…Domain 1 is located on the other side of the arched gap and binds to the N-terminal peptide of syntaxin-1 ( Fig. 1 ) ( 16 ).…”