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The accessory helix of complexin functions by stabilizing central helix secondary structure.

ABSTRACT: The presynaptic protein complexin (CPX) is a critical regulator of synaptic vesicle fusion, but the mechanisms underlying its regulatory effects are not well understood. Its highly conserved central helix (CH) directly binds the ternary SNARE complex and is required for all known CPX functions. The adjacent accessory helix (AH) is not conserved despite also playing an important role in CPX function, and numerous models for its mechanism have been proposed. We examined the impact of AH mutations and chimeras on CPX function in vivo and in vitro using C. elegans. The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved. CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function. We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions.

SUBMITTER: Radoff DT

PROVIDER: S-EPMC4270070 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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The accessory helix of complexin functions by stabilizing central helix secondary structure.

Radoff Daniel T DT Dong Yongming Y Snead David D Bai Jihong J Eliezer David D Dittman Jeremy S JS

eLife 20141110

The presynaptic protein complexin (CPX) is a critical regulator of synaptic vesicle fusion, but the mechanisms underlying its regulatory effects are not well understood. Its highly conserved central helix (CH) directly binds the ternary SNARE complex and is required for all known CPX functions. The adjacent accessory helix (AH) is not conserved despite also playing an important role in CPX function, and numerous models for its mechanism have been proposed. We examined the impact of AH mutations ...[more]

PMID: 25383924

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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

The accessory helix of complexin functions by stabilizing central helix secondary structure.

Publications

The accessory helix of complexin functions by stabilizing central helix secondary structure.

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