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.

Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo

“…However, we cannot rule out that these mutants may also disrupt a distinct in vivo Cpx-SNARE interaction beyond the predicted trans Cpx/SNARE array. Indeed, there is evidence that mutations within the N-terminal domain of Cpx may alter release by blocking cis-SNARE zippering as well (34). Unlike NC mCpx mutants, we observed a strong effect of the SC mCpx mutants, which enhanced the clamping function of Cpx in vivo, and enhanced formation of trans interactions in vitro (26).…”

Transmission, Development, and Plasticity of Synapses

“…Loss of cpx function results in two distinct phenotypes at Drosophila NMJs: a dramatic increase in spontaneous release and a reduction in evoked release (Huntwork and Littleton 2007;Xue et al 2009;Cho et al 2010;Jorquera et al 2012;Buhl et al 2013;Bykhovskaia et al 2013;Iyer et al 2013;Choi et al 2014). These findings suggest a dual role for Cpx at the synapse.…”

“…Syt1 lacking C2B Ca 2+ binding shows a dramatic reduction in synchronous release, but is capable of preventing asynchronous fusion. (C) Model of complexin regulation of SNARE assembly (modified from Bykhovskaia et al 2013). Cpx (magenta) stabilizes a partially zippered SNARE bundle, leading to separation of the vesicle and plasma membranes (right).…”

“…Null mutations in Snap-25 have relatively normal neurotransmitter release at third instar NMJs, with the similar SNAP-24 protein proposed to compensate for the loss (Vilinsky et al 2002). These redundancy issues confound robust structure-function approaches that have been applied to other proteins like Synaptotagmin, although there have been some efforts in the field to examine hypomorphic mutations of the SNARE proteins in Drosophila Wu et al 1999;Stewart et al 2000;Rao et al 2001b;Bykhovskaia et al 2013;Megighian et al 2013;Demill et al 2014). Although the requirement for the SNARE complex in fusion is well accepted, there are still numerous questions.…”

“…A popular model is that Cpx binds to SNARE complexes during zippering, preventing full assembly prior to fusion ( Figure 2C). There are computational studies to support such a role (Bykhovskaia et al 2013), but it still remains speculative at this point. Another potential mechanism for Cpx as a clamp is suggested by the suppression of the dramatic increase in minis (.50-fold in cpx mutants alone) in double mutants with syt1 ( Figure 2D) .…”