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Binding of Munc18-1 to synaptobrevin and to the SNARE four-helix bundle.

ABSTRACT: Sec1/Munc18 (SM) proteins and soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form part of the core intracellular membrane fusion machinery, but it is unclear how they cooperate in membrane fusion. The synaptic vesicle SNARE synaptobrevin and the plasma membrane SNAREs syntaxin-1 and SNAP-25 assemble into a tight SNARE complex that includes a four-helix bundle formed by their SNARE motifs and is key for fusion. The neuronal SM protein Munc18-1 binds to syntaxin-1 and to the SNARE complex through interactions with the syntaxin-1 N-terminal region that are critical for neurotransmitter release. It has been proposed that Munc18-1 also binds to synaptobrevin and to the SNARE four-helix bundle and that such interactions might be crucial for membrane fusion, but definitive, direct evidence of these interactions has not been described. Using diverse biophysical approaches, we now demonstrate that Munc18-1 indeed binds to synaptobrevin and to the SNARE four-helix bundle. Both interactions have similar affinities (in the low micromolar range) and appear to involve the same cavity of Munc18-1 that binds to syntaxin-1. Correspondingly, the N-terminal region of syntaxin-1 competes with the SNARE four-helix bundle and synaptobrevin for Munc18-1 binding. Importantly, the Munc18-1 binding site on synaptobrevin is located at the C-terminus of its SNARE motif, suggesting that this interaction places Munc18-1 right at the site where fusion occurs. These results suggest a model in which neurotransmitter release involves a sequence of three different types of Munc18-1-SNARE interactions and in which Munc18-1 plays a direct, active role in membrane fusion in cooperation with the SNAREs.

SUBMITTER: Xu Y

PROVIDER: S-EPMC2834481 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Binding of Munc18-1 to synaptobrevin and to the SNARE four-helix bundle.

Xu Yi Y Su Lijing L Rizo Josep J

Biochemistry 20100301 8

Sec1/Munc18 (SM) proteins and soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form part of the core intracellular membrane fusion machinery, but it is unclear how they cooperate in membrane fusion. The synaptic vesicle SNARE synaptobrevin and the plasma membrane SNAREs syntaxin-1 and SNAP-25 assemble into a tight SNARE complex that includes a four-helix bundle formed by their SNARE motifs and is key for fusion. The neuronal SM protein Munc18-1 binds to syntaxin-1 ...[more]

PMID: 20102228

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Project description:The four-alpha-helix bundle mimics the transmembrane domain of the Cys-loop receptor family believed to be the protein target for general anesthetics. Using high resolution NMR, we solved the structure (Protein Data Bank ID: 2I7U) of a prototypical dimeric four-alpha-helix bundle, (Aalpha(2)-L1M/L38M)(2,) with designed specific binding pockets for volatile anesthetics. Two monomers of the helix-turn-helix motif form an antiparallel dimer as originally designed, but the high-resolution structure exhibits an asymmetric quaternary arrangement of the four helices. The two helices from the N-terminus to the linker (helices 1 and 1') are associated with each other in the dimer by the side-chain ring stacking of F12 and W15 along the long hydrophobic core and by a nearly perfect stretch of hydrophobic interactions between the complementary pairs of L4, L11, L18, and L25, all of which are located at the heptad e position along the helix-helix dimer interface. In comparison, the axes of the two helices from the linker to the C-terminus (helices 2 and 2') are wider apart from each other, creating a lateral access pathway around K47 from the aqueous phase to the center of the designed hydrophobic core. The site of the L38M mutation, which was previously shown to increase the halothane binding affinity by approximately 3.5-fold, is not part of the hydrophobic core presumably involved in the anesthetic binding but shows an elevated transverse relaxation (R(2)) rate. Qualitative analysis of the protein dynamics by reduced spectral density mapping revealed exchange contributions to the relaxation at many residues in the helices. This observation was confirmed by the quantitative analysis using the Modelfree approach and by the NMR relaxation dispersion measurements. The NMR structures and Autodock analysis suggest that the pocket with the most favorable amphipathic property for anesthetic binding is located between the W15 side chains at the center of the dimeric hydrophobic core, with the possibility of two additional minor binding sites between the F12 and F52 ring stacks of each monomer. The high-resolution structure of the designed anesthetic-binding protein offers unprecedented atomistic details about possible sites for anesthetic-protein interactions that are essential to the understanding of molecular mechanisms of general anesthesia.

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Binding of Munc18-1 to synaptobrevin and to the SNARE four-helix bundle.

Publications

Binding of Munc18-1 to synaptobrevin and to the SNARE four-helix bundle.

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