Evolutionary Divergence of the C-terminal Domain of Complexin Accounts for Functional Disparities between Vertebrate and Invertebrate Complexins.

Rachel T Wragg,Ishani Basu,Harel Weinstein,David Eliezer,Daniel A Parisotto,Jeremy S Dittman,Zhenlong Li,David Snead,Mayu S Terakawa

doi:10.3389/fnmol.2017.00146

Abstract

Complexin is a critical presynaptic protein that regulates both spontaneous and calcium-triggered neurotransmitter release in all synapses. Although the SNARE-binding central helix of complexin is highly conserved and required for all known complexin functions, the remainder of the protein has profoundly diverged across the animal kingdom. Striking disparities in complexin inhibitory activity are observed between vertebrate and invertebrate complexins but little is known about the source of these differences or their relevance to the underlying mechanism of complexin regulation. We found that mouse complexin 1 (mCpx1) failed to inhibit neurotransmitter secretion in Caenorhabditis elegans neuromuscular junctions lacking the worm complexin 1 (CPX-1). This lack of inhibition stemmed from differences in the C-terminal domain (CTD) of mCpx1. Previous studies revealed that the CTD selectively binds to highly curved membranes and directs complexin to synaptic vesicles. Although mouse and worm complexin have similar lipid binding affinity, their last few amino acids differ in both hydrophobicity and in lipid binding conformation, and these differences strongly impacted CPX-1 inhibitory function. Moreover, function was not maintained if a critical amphipathic helix in the worm CPX-1 CTD was replaced with the corresponding mCpx1 amphipathic helix. Invertebrate complexins generally shared more C-terminal similarity with vertebrate complexin 3 and 4 isoforms, and the amphipathic region of mouse complexin 3 significantly restored inhibitory function to worm CPX-1. We hypothesize that the CTD of complexin is essential in conferring an inhibitory function to complexin, and that this inhibitory activity has been attenuated in the vertebrate complexin 1 and 2 isoforms. Thus, evolutionary changes in the complexin CTD differentially shape its synaptic role across phylogeny.

Highlights

Precise synaptic transmission is critical for proper nervous system function, and over the past 25 years, most of the proteins required for this process have been identified and characterized
Several crystal structures of complexin bound to the ternary SNARE complex as well as biochemical and in vivo studies have identified specific residues required for the tight association of the central helix (CH) and the ternary SNARE bundle, and these residues are almost perfectly conserved throughout phylogeny (Nonet et al, 1998; Saifee et al, 1998; Bracher et al, 2002; Chen et al, 2002; Giraudo et al, 2006; Xue et al, 2007; Maximov et al, 2009) (Figure 1D)
In the experiments described here, several features of complexin conservation were explored in the context of in vivo synaptic function using a simple behavioral assay as well as in vitro membrane binding assays

Summary

Introduction

Precise synaptic transmission is critical for proper nervous system function, and over the past 25 years, most of the proteins required for this process have been identified and characterized. The complete molecular picture of the events controlling SV fusion is far from fully developed, there is general agreement on the impact of perturbing the SNAREs, Munc, Munc, and synaptotagmin in many highly divergent experimental preparations such as the squid giant synapse, worm and fly neuromuscular junctions, as well as rodent cultured neurons and acute rodent brain slices (Augustine et al, 1996; Weimer and Jorgensen, 2003; Sudhof and Rothman, 2009; Kochubey et al, 2011; Sudhof and Rizo, 2011) Consensus of both sequence and function across such a broad range of synapses implies a deep mechanistic conservation of calciumregulated secretion in all animals, consistent with the assertion that there is a single overarching molecular pathway for SV fusion in neurons shared across phylogeny. These observations hint at a transient role for mammalian complexin 1 in preventing premature fusion during the process of docking and priming, whereas invertebrate complexins are constitutively required to inhibit spontaneous fusion

Methods

Results

Conclusion