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

Daniel T Radoff,David Eliezer,Yongming Dong,David Snead,Jihong Bai,Jeremy S Dittman

doi:10.7554/elife.04553

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.

Highlights

Precise control of synaptic vesicle fusion at the presynaptic terminal endows a nervous system with the means to regulate functional synaptic connectivity
This so-called ‘central helix’ (CH) is deeply conserved across phylogeny (76% identity between mammals and nematodes), whereas the adjacent helical sequence corresponding to the mouse accessory helix (AH) is much more heterogeneous, with only 20% identity between mammals and nematodes based on the 18 residues N-terminal to CH (Figure 1B)
The stable helical structure of the AH domain is predicted to be deeply conserved across phylogeny based on the analysis of complexin sequences in 16 diverse metazoan species from seven phyla ranging from Trychoplax to human (Figure 1—figure supplements 1–2)

Summary

Introduction

Precise control of synaptic vesicle fusion at the presynaptic terminal endows a nervous system with the means to regulate functional synaptic connectivity. The core machinery of the fusion process is composed of the neuronal SNAREs (VAMP2, syntaxin 1, and SNAP-25) along with several SNARE-binding proteins such as synaptotagmin, Munc, and Munc (Sudhof and Rothman, 2009; Sudhof, 2013) These proteins are highly similar in sequence and function across species, reflecting the deep conservation of synaptic transmission. Mammals possess four isoforms of complexin, and deletion of the two broadly expressed isoforms (mCpx1/2) is lethal (Reim et al, 2001; Xue et al, 2008) This small cytoplasmic protein possesses an alpha-helical SNARE-binding domain known as the central helix (CH), which is broadly conserved among metazoa (Pabst et al, 2000; Reim et al, 2001; Bracher et al, 2002; Chen et al, 2002; Brose, 2008). A wide variety of models for AH function have been proposed including direct binding to SNAREs or other proteins

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