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

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

Problem Statement
While the complement regulatory protein CD59 has been identified as a promoter of SNARE complex assembly and synaptic vesicle fusion, the specific molecular mechanisms mediating this interaction remain poorly characterized. Specifically, the key domains within the vesicle-associated membrane protein 2 (VAMP2) that facilitate binding to CD59, and the structural determinants within CD59 required for this function, have not been systematically defined. Furthermore, it is unclear whether specific point mutations in CD59, which are known to affect its classical complement-inhibitory functions, impact its ability to regulate SNARE complex assembly.

Methodology
The study employed a multidisciplinary approach combining experimental biochemistry, cell biology, and computational modeling:

• Cellular and Biochemical Assays: Co-immunoprecipitation (co-IP) and immunofluorescence confocal microscopy were used to validate the physical interaction and spatial colocalization of CD59 and VAMP2 in HEK293T and SH-SY5Y cells. Western blotting under non-boiled and boiled conditions assessed SNARE complex assembly and individual protein expression levels.
• Mutagenesis: Four single-point mutants of CD59 (N18Q, W40E, C64T, N77A) were generated to test the impact of specific residues on function. Four domain-deletion mutants of VAMP2 (ΔP-rich NT, ΔSNARE motif, ΔJMD, ΔTMD) were constructed to map the binding interface.
• Computational Modeling:
  • AlphaFold3: Used to predict interaction structures between CD59 and full-length VAMP2, as well as individual VAMP2 domains. Confidence was assessed using a combined ipTM + pTM threshold of ≥ 0.75.
  • Molecular Dynamics (MD) Simulations: 100 ns simulations were conducted using GROMACS to evaluate the stability (RMSD, Radius of Gyration) and interaction features (hydrogen bond counts) of CD59 (wild-type and mutants) bound to VAMP2 and the pre-assembled SNARE complex.

Key Results

1. Confirmation of Interaction and Function: CD59 was confirmed to directly bind VAMP2 and promote the assembly of the SNARE complex (comprising Syntaxin-1A, SNAP-25, and VAMP2) in a dose-dependent manner. This promotion occurred without altering the expression levels of the individual SNARE proteins.
2. Resilience of CD59 Point Mutants: Despite molecular dynamics simulations indicating differential effects on binding stability (e.g., W40E showed reduced stability, while C64T and N77A showed increased stability), all four CD59 point mutants retained the ability to promote SNARE complex assembly to a level comparable to wild-type CD59.
3. Mapping the Binding Interface:
   • AlphaFold3 Limitations: Prediction of the full-length CD59–VAMP2 complex yielded a low confidence score (0.48), likely due to the flexibility of full-length proteins.
   • Domain-Specific Prediction: When individual VAMP2 domains were analyzed, high-confidence interactions (ipTM + pTM ≥ 0.75) were predicted for the Proline-rich N-terminal (P-rich NT) domain, the Juxtamembrane domain (JMD), and the Transmembrane domain (TMD). The SNARE motif yielded a score below the threshold.
   • Experimental Validation: Co-IP assays with VAMP2 truncation mutants confirmed that deletion of the P-rich NT domain abolished the interaction with CD59, whereas deletion of the SNARE motif did not. MD simulations supported this, showing that the ΔP-rich NT complex was the least stable, while the ΔSNARE motif complex remained stable.
4. Structural Dynamics: MD simulations revealed that the ΔSNARE motif truncation formed the most compact and stable complex with CD59 but with fewer hydrogen bonds, whereas the ΔP-rich NT truncation exhibited high conformational fluctuation and a dense but likely transient hydrogen bond network.

Key Contributions

• Identification of the Binding Interface: The study identifies the P-rich N-terminal domain of VAMP2 as the critical mediator for CD59 interaction, distinguishing it from the core SNARE motif.
• Functional Robustness: The findings demonstrate that the SNARE assembly-promoting function of CD59 is robust to specific point mutations (N18Q, W40E, C64T, N77A) that alter its binding energetics or complement inhibitory activity.
• Methodological Insight: The paper highlights the utility of combining AlphaFold3 with domain-specific inputs and MD simulations to resolve protein-protein interactions where full-length predictions fail due to intrinsic disorder or flexibility.

Significance
The paper establishes that CD59 promotes SNARE complex assembly by binding to the P-rich N-terminal domain of VAMP2 rather than the SNARE motif itself. This binding mode suggests a chaperone-like role where CD59 facilitates assembly without occupying the core interface required for SNARE zippering. The study concludes that this regulatory mechanism possesses functional robustness, as the examined point mutations do not impair the assembly-promoting function. These findings provide a structural basis for understanding the non-canonical synaptic functions of CD59 and suggest that this interaction is distinct from its classical role in inhibiting the membrane attack complex. The authors note that while the study identifies key domains, further structural biology (e.g., NMR, cryo-EM) and live-cell imaging are required to fully resolve the dynamic interaction mechanism and the influence of lipid raft microenvironments.