Phosphorylation of SNAP-25 at Ser187 is enhanced following its cleavage by Botulinum Neurotoxin Serotype A, promoting the dominant-negative effect of the resulting fragment

Date

2025-10-01

Author

Koc, Dilara
Ezgin, Sena
Kavakli, Ebru
Kota, Krishna P.
Sen, Edanur
Mahone, Christopher
Palko, Mary Ellen
Cazares, Lisa H.
Can, Tolga
Tessarollo, Lino
Bavari, Sina
Marıon, Antoıne
Kiriş, Erkan

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Botulinum Neurotoxin Serotype A (BoNT/A), responsible for most human botulism cases, inhibits neurotransmitter release by cleaving the target protein SNAP-25. Previous literature demonstrated that BoNT/A mediated cleavage of a small subset of the SNAP-25 pool, resulting in SNAP-25 (1–197) fragments, is sufficient to block exocytosis. SNAP-25 (1–197) potentially competes against intact SNAP-25 for SNARE complexes and blocks neurotransmission through a dominant-negative mechanism. However, how a tiny fraction of cleaved SNAP-25 efficiently outcompetes a large pool of intact SNAP-25 remains unknown. Here, we examined the importance of SNAP-25 phosphorylation at Ser187 residue, located in the C-terminus SNARE domain, in the context of BoNT action. Our results demonstrated that Ser187-phosphorylated SNAP-25 can be efficiently cleaved in cells. Importantly, BoNT/A-cleaved SNAP-25 fragments in neuronal and non-neuronal cells are heavily phosphorylated at Ser187 and localized on the cell membrane. SNAP-25 (1–197) binds to syntaxin-1A, and the interaction is enhanced by Ser187 phosphorylation. We also found that SNAP-25 (1–197) survives longer than the BoNT/A enzymatic component itself in cells. Molecular modeling suggested that SNAP-25 (1–197), phosphorylated or not, forms stable SNARE complexes; however, Ser187 phosphorylation induces local changes in surface electrostatic potential and dynamics of the complex. This study characterizes the molecular mechanism underlying the dominant-negative effect of SNAP-25 (1–197) on neurotransmission. This research could have implications for the future development of BoNT/A inhibitors and the generation of new BoNT/A clinical formulations by regulating the abundance of Ser187 phosphorylation in cleaved SNAP-25 fragments.

URI

https://hdl.handle.net/11511/116598

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Department of Biology, Article