How disease mutations disrupt the protein machine that enables neurotransmitter release

The SNARE Complex and its assembly

Fast and reliable neurotransmission at chemical synapses depends on the precise exocytotic fusion of synaptic vesicles at the presynaptic membrane, enabling the release of neurotransmitters. This fusion is driven by the assembly of the SNARE complex, a four-helix bundle composed of Syntaxin-1 (Stx1), Synaptobrevin-2 (Syb2, also known as VAMP2), and SNAP‑25, which fuses the vesicle and presynaptic membranes through a zippering mechanism. The formation of the SNARE complex (Figure 1) is a multistep, tightly regulated process coordinated by regulatory proteins such as Munc18-1 (also known as STXBP1) and Munc13-1. Because the SNARE assembly depends on transient intermediates and finely tuned protein–protein interfaces, genetic variants can perturb specific steps of the pathway and compromise synaptic release.

Figure 1. Stepwise assembly of the neuronal SNARE complex during synaptic vesicle fusion. Schematic view of the SNARE complex formation as a multistep pathway coordinated by regulatory factors. Vesicle tethering by Munc13-1 and initial engagement of Syb2 proceed through formation of a templated intermediate with Munc18-1:Stx1, followed by a half-zippered SNARE state with incorporated SNAP-25 and completion of zippering into the fully assembled SNARE complex that drives membrane fusion. 

The SNAREopathy genotype–phenotype landscape

Mutations in SNARE complex components may lead to a range of severe neurological disorders collectively referred to as SNAREopathies (Figure 2, Ref [1]). Mutations in core SNARE proteins and key regulators, including Stx1B, SNAP-25, Syb2, Munc18-1, and Munc13-1, are associated with overlapping clinical features such as seizures, abnormal EEG patterns, developmental delay, speech impairment, and movement or autism-spectrum phenotypes[2]. Yet, despite this growing catalogue of disease variants, the exact molecular mechanisms remain unclear: different mutations may derail distinct interactions or trap the pathway in different assembly intermediates.

Figure 2. Different synaptic proteins are linked to different neurological disorders. Links between mutations in SNARE machinery components and clinical features (based on Ref [1]).

Research focus

This project focuses on examining how SNAREopathic mutations affect the molecular mechanism of SNARE complex assembly by structurally characterizing key protein–protein interactions. SNAREopathic mutations and other functional mutations also act as mechanistic probes to understand the interactions between core SNARE components and their regulatory partners. In doing so, we aim to connect specific molecular defects in SNARE machinery to impaired synaptic vesicle fusion and neurological disease.

Our approach

Building up on our earlier work[3], we combine complementary structural and biophysical methods to investigate the SNARE assembly pathway in both wild type and SNAREopathic mutant backgrounds. Crystallography and Cryo-electron microscopy (Cryo-EM) provide structural snapshots of key intermediates and regulatory complexes, while nuclear magnetic resonance (NMR)-spectroscopy resolves conformational dynamics and transient or weak interactions that dominate early assembly steps. Cross-linking mass spectrometry (XL-MS) supplies interaction/contact maps across heterogeneous states and conditions, supporting comparisons of how variants rewire proximity networks. Finally, isothermal titration calorimetry (ITC) quantifies binding affinities, enabling us to connect mutation-induced structural or dynamic changes to altered interaction strength along the assembly route.

Researchers

●      Dr. Xiao Jakob Schmitt — Postdoctoral researcher

●      Helena Rheinbay — PhD student 

Collaborators

This project benefits from several international collaborations. Our collaboration with the Krogan Group at University California San Francisco (UCSF) focuses on the SNAREopathic mutations in neuronal cell lines. Furthermore, we collaborate with Dr. Camila Esguerra and Dr. Wietske van der Ent (University of Oslo), as well as with the group of Prof. Sigrist (Freie Universität Berlin); these partners are studying disease-relevant mutations in in-vivo models of Zebrafish and Drosophila, respectively.

Funding

We acknowledge the Einstein Foundation for financing the project. 

References

[1] Verhage, M., Sørensen, J. B. SNAREopathies: Diversity in Mechanisms and Symptoms. Neuron 107(1), 22–37 (2020). https://doi.org/10.1016/j.neuron.2020.05.036

[2] Vardar, G., Gerth, F., Schmitt, X. J., Rautenstrauch, P., Trimbuch, T., Schubert, J., Lerche, H., Rosenmund, C., Freund, C. Epilepsy-causing STX1B mutations translate altered protein functions into distinct phenotypes in mouse neurons. Brain 143(7), 2119–2138 (2020). https://doi.org/10.1093/brain/awaa151

[3] Schmitt, X. J. (2024). Molecular analysis of the SNARE complex formation machinery and the impact of SNAREopathic mutations (dissertation). Freie Universität Berlin. URL: https://refubium.fu-berlin.de/bitstream/handle/fub188/46055/Dissertation_Xiao_Jakob_Schmitt-1.pdf?sequence=4&isAllowed=y