CD59 Promotes SNARE Complex Assembly via Interaction with the Proline-Rich N-Terminal Domain of VAMP2

Authors

Xiang, H., Liu, Y., Feng, J., Wen, W., Wen, L.

Abstract

Objective: The complement regulatory protein CD59 has been shown to promote SNARE complex assembly, yet its interaction with vesicle-associated membrane protein 2 (VAMP2) remains poorly characterized. This study aims to identify the key domain in VAMP2 that mediates the CD59 interaction and to evaluate whether CD59 point mutations affect SNARE complex assembly. Methods: The interaction between CD59 and VAMP2 was examined by immunofluorescence confocal microscopy and co-immunoprecipitation (co-IP). The effect of CD59 on SNARE complex assembly was assessed by co-expressing CD59 with the three core SNARE proteins (syntaxin-1, SNAP-25, and VAMP2) and detecting complex formation by western blotting. Four CD59 single-point mutants were generated and evaluated in SNARE assembly assays. AlphaFold3 was employed to predict the interaction between CD59 and individual VAMP2 domains (confidence threshold: ipTM + pTM >= 0.75). Truncated VAMP2 constructs were further characterized by molecular dynamics simulations and co-IP. Results: (1) CD59 directly bound VAMP2 and promoted SNARE complex assembly without altering individual SNARE protein levels. (2) All four CD59 single-point mutants retained the ability to promote SNARE assembly at a level comparable to wild-type CD59, despite showing differential effects on binding stability in molecular dynamics simulations. (3) The proline-rich (P-rich) N-terminal domain of VAMP2 was identified as the key binding interface; its deletion abolished the CD59 interaction, whereas deletion of the SNARE motif did not. Conclusion: CD59 promotes SNARE complex assembly through interaction with the P-rich N-terminal domain of VAMP2. The examined point mutations do not impair this function, suggesting functional redundancy in the contact residues that maintain the interaction.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 07 Aug 2026.

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