Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly

ABSTRACTSynaptic vesicle fusion is mediated by membrane-bridging complexes formed by SNARE proteins - VAMP2 on the vesicle and Syntaxin-1/SNAP25 on the pre-synaptic membrane. Accumulating evidence suggest that chaperones Munc18-1 and Munc13-1 co-operatively catalyze SNARE assembly via an intermediate ‘template’ complex containing Syntaxin-1 and VAMP2. How SNAP25 is chaperoned into this nascent complex remains a mystery. Here we report that Munc13-1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single-molecule optical tweezer studies. Detailed structure-function analyses show that the binding is mediated by the Munc13-1 MUN domain and is specific for the SNAP25 ‘linker’ region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and presumably bound in ~1:1 stoichiometry by the self-assembled Munc13-1 nanoclusters. Our data suggests that Munc13-1’s capacity to bind all three synaptic SNARE proteins likely underlie its chaperone function.

The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation

Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation

ABSTRACTFusion of intracellular trafficking vesicles is mediated by the assembly of soluble N-ethylmaleimide-sensitive fusion protein receptors (SNAREs) to form membrane-bridging complexes. Also required for SNARE-mediated membrane fusion are Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. In the paradigmatic structure of an SM–SNARE complex, Munc18-1 bound to the Qa-SNARE syntaxin 1, the SNARE protein is trapped in an autoinhibited closed conformation that prevents it from entering into SNARE complexes. Here, we present the structure of a second SM–Qa-SNARE complex, Vps45–Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its three-helical Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Tlg2 has a pronounced tendency to self-associate via its SNARE motif, and we demonstrate that Vps45 can rescue Tlg2 oligomers into stoichiometric Vps45–Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

Munc13-1 MUN domain and Munc18-1 cooperatively chaperone SNARE assembly through a tetrameric complex

Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins—syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.

Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. Next, SNAP-25 binds the templated SNAREs to form a partially-zippered SNARE complex. Finally, full zippering displaces Munc18-1. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

Synthesis, structure, and in vitro anti-lung cancer activity on an In-based nanoscale coordination polymer

Here, a new indium (In)-based coordination polymer [In(hip)](DMF)2(H2O)3 (1, DMF=N,N-dimethylformamide) was successfully prepared by a solvothermal reaction of In(NO3)3·6H2O and 5-hydroxyisophthalic acid (H3hip) in a mixed solvent of DMF and H2O with the presence of NaCl as a template. Complex 1 was characterized by elemental analysis (EA), single-crystal X-ray crystallography, and powder X-ray diffraction (PXRD), and the results reveal that complex 1 shows a two-dimensional (2D) grid-like network with considerable solvent accessible volume that was generated from the packing of the 2D layers via the AB pattern. Furthermore, complex 1 could be downsized into nanoscale particles with the aid of polyvinylpyrrolidone (PVP). In addition, the anticancer activities of 1 and the nanoscale 1 were probed via the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay.

Insight into the mechanism of nonenzymatic RNA primer extension from the structure of an RNA-GpppG complex

The nonenzymatic copying of RNA templates with imidazole-activated nucleotides is a well-studied model for the emergence of RNA self-replication during the origin of life. We have recently discovered that this reaction can proceed through the formation of an imidazolium-bridged dinucleotide intermediate that reacts rapidly with the primer. To gain insight into the relationship between the structure of this intermediate and its reactivity, we cocrystallized an RNA primer–template complex with a close analog of the intermediate, the triphosphate-bridged guanosine dinucleotide GpppG, and solved a high-resolution X-ray structure of the complex. The structure shows that GpppG binds the RNA template through two Watson–Crick base pairs, with the primer 3ʹ-hydroxyl oriented to attack the 5ʹ-phosphate of the adjacent G residue. Thus, the GpppG structure suggests that the bound imidazolium-bridged dinucleotide intermediate would be preorganized to react with the primer by in-line SN2 substitution. The structures of bound GppG and GppppG suggest that the length and flexibility of the 5ʹ-5ʹ linkage are important for optimal preorganization of the complex, whereas the position of the 5ʹ-phosphate of bound pGpG explains the slow rate of oligonucleotide ligation reactions. Our studies provide a structural interpretation for the observed reactivity of the imidazolium-bridged dinucleotide intermediate in nonenzymatic RNA primer extension.