Dataset Information

Interaction of the complexin accessory helix with the C-terminus of the SNARE complex: molecular-dynamics model of the fusion clamp.

ABSTRACT: SNARE complexes form between the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 to drive membrane fusion. A cytosolic protein, complexin (Cpx), binds to the SNARE bundle, and its accessory helix (AH) functions to clamp synaptic vesicle fusion. We performed molecular-dynamics simulations of the SNARE/Cpx complex and discovered that at equilibrium the Cpx AH forms tight links with both synaptobrevin and SNAP25. To simulate the effect of electrostatic repulsion between vesicle and membrane on the SNARE complex, we calculated the electrostatic force and performed simulations with an external force applied to synaptobrevin. We found that the partially unzipped state of the SNARE bundle can be stabilized by interactions with the Cpx AH, suggesting a simple mechanistic explanation for the role of Cpx in fusion clamping. To test this model, we performed experimental and computational characterizations of the syx(3-69)Drosophila mutant, which has a point mutation in syntaxin that causes increased spontaneous fusion. We found that this mutation disrupts the interaction of the Cpx AH with synaptobrevin, partially imitating the cpx null phenotype. Our results support a model in which the Cpx AH clamps fusion by binding to the synaptobrevin C-terminus, thus preventing full SNARE zippering.

SUBMITTER: Bykhovskaia M

PROVIDER: S-EPMC3736676 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Interaction of the complexin accessory helix with the C-terminus of the SNARE complex: molecular-dynamics model of the fusion clamp.

Bykhovskaia Maria M Jagota Anand A Gonzalez Agustin A Vasin Alexander A Littleton J Troy JT

Biophysical journal 20130801 3

SNARE complexes form between the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 to drive membrane fusion. A cytosolic protein, complexin (Cpx), binds to the SNARE bundle, and its accessory helix (AH) functions to clamp synaptic vesicle fusion. We performed molecular-dynamics simulations of the SNARE/Cpx complex and discovered that at equilibrium the Cpx AH forms tight links with both synaptobrevin and SNAP25. To simulate the effect of electrostatic re ...[more]

PMID: 23931316

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Project description:Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the plasma membrane. Vesicles attach to membranes via a specialized protein machinery composed of membrane-attached (t-SNARE) and vesicle-attached (v-SNARE) proteins that zipper together to form a coiled-coil SNARE bundle that brings the two fusing membranes into close proximity. Neurotransmitter release may occur either in response to an action potential or through spontaneous fusion. A cytosolic protein, Complexin (Cpx), binds the SNARE complex and restricts spontaneous exocytosis by acting as a fusion clamp. We previously proposed a model in which the interaction between Cpx and the v-SNARE serves as a spring to prevent premature zippering of the SNARE complex, thereby reducing the likelihood of fusion. To test this model, we combined molecular-dynamics (MD) simulations and site-directed mutagenesis of Cpx and SNAREs in Drosophila. MD simulations of the Drosophila Cpx-SNARE complex demonstrated that Cpx's interaction with the v-SNARE promotes unraveling of the v-SNARE off the core SNARE bundle. We investigated clamping properties in the syx3-69 paralytic mutant, which has a single-point mutation in the t-SNARE and displays enhanced spontaneous release. MD simulations demonstrated an altered interaction of Cpx with the SNARE bundle that hindered v-SNARE unraveling by Cpx, thus compromising clamping. We used our model to predict mutations that should enhance the ability of Cpx to prevent full assembly of the SNARE complex. MD simulations predicted that a weakened interaction between the Cpx accessory helix and the v-SNARE would enhance Cpx flexibility and thus promote separation of SNAREs, reducing spontaneous fusion. We generated transgenic Drosophila with mutations in Cpx and the v-SNARE that disrupted a salt bridge between these two proteins. As predicted, both lines demonstrated a selective inhibition in spontaneous release, suggesting that Cpx acts as a fusion clamp that restricts full SNARE zippering.

Dataset Information

Interaction of the complexin accessory helix with the C-terminus of the SNARE complex: molecular-dynamics model of the fusion clamp.

Publications

Interaction of the complexin accessory helix with the C-terminus of the SNARE complex: molecular-dynamics model of the fusion clamp.

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