The SNARE complex plays a central role in synaptic vesicle fusion, forming a four-helix bundle between synaptobrevin on the vesicle membrane and syntaxin and SNAP25 on the plasma membrane. This assembly drives membrane fusion and neurotransmitter release. Complexin (Cpx), a small cytosolic protein, binds to the SNARE complex and functions as a fusion clamp, preventing spontaneous fusion while facilitating evoked release. Despite extensive research, the precise molecular mechanism by which Cpx clamps fusion remains unclear. In this study, we employed molecular dynamics (MD) simulations to investigate the interaction between the Cpx accessory helix (AH) and the C-terminus of the SNARE complex, aiming to elucidate how Cpx stabilizes a partially unzipped state that acts as a fusion clamp.
We began by constructing an initial model of the SNARE/Cpx complex using high-resolution X-ray structures: 1N7S for the SNARE complex and 1KIL for the SNARE/Cpx complex. The initial topology was optimized using Monte-Carlo minimization (MCM) with the ZMM/MVM software package. Subsequent MD simulations were performed using NAMD and VMD, employing the CHARMM22 force field with CMAP correction. A water box with KCl at 150 mM concentration was used to mimic physiological conditions. Simulations were conducted under periodic boundary conditions with Ewald electrostatics and Langevin thermostat at 300 K.
Our results revealed that in a solvated environment, the Cpx AH forms tight, stable interactions with both synaptobrevin (Syb) and the C-terminal domain of SNAP25 (SN2). These interactions are stabilized by salt bridges and hydrophobic contacts, particularly involving residues K37 and L41 of Cpx. Over a 250 ns simulation, the Cpx AH transitioned from transient contact with Syb to a stable position within the groove between Syb and SN2, where it remained for the duration of the simulation despite brief disruptions of individual salt bridges.
To assess the impact of membrane repulsion on SNARE stability, we calculated the electrostatic repulsive force between the vesicle and plasma membranes. Using the Debye-Hückel approximation, we estimated forces ranging from 93 pN (fixed potential) to 210 pN (fixed charge), depending on assumptions about surface charge regulation. Applying an external force of 140 pN (2 kcal/mol/Å) to the C-terminal residue of Syb (W89) induced rapid separation of layers 8 and partial disruption of layer 7, but not layer 6. The energy barrier for unzipping was modest, suggesting that such a partially unzipped state is energetically accessible. However, further unzipping beyond layer 7 required significantly higher forces, which are unlikely to be generated by membrane repulsion alone due to its steep distance dependence.
When Cpx was present, the partially unzipped state became more stable.Pyridostigmine medchemexpress Relaxation simulations showed that in the presence of Cpx, the SNARE complex maintained a separated conformation with layers 7 and 8 unzipped, and the energy declined consistently below baseline.DOTAP NF-κB In contrast, without Cpx, the complex tended to rezip.PMID:34972260 This indicates that Cpx stabilizes a metastable, partially unzipped state—consistent with a fusion clamp.
We tested this model using the Drosophila syx3-69 mutant, which exhibits enhanced spontaneous release resembling the cpx null phenotype. The T251I mutation in syntaxin alters the local structure of layer 7, disrupting the interaction between the Cpx AH and Syb. MD simulations confirmed that this mutation shifts Cpx’s orientation toward SN2, weakening its binding to Syb. Experimentally, focal recordings from neuromuscular junctions showed increased spontaneous release in syx3-69, though less severe than in cpx null mutants—supporting a partial loss-of-function phenotype.
In conclusion, our computational and experimental data support a model in which the Cpx AH clamps fusion by binding to the C-terminus of Syb, stabilizing a partially unzipped SNARE complex. This prevents full zippering and maintains the vesicle at a distance (~5 nm) where fusion cannot occur. The model explains both the clamping function of Cpx and the phenotypic effects of mutations like syx3-69, providing a mechanistically coherent and energetically favorable explanation for synaptic fusion control.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com