Rabphilin 3A binds the N-peptide of SNAP-25 to promote SNARE complex assembly in exocytosis

Abstract

Exocytosis of secretory vesicles requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and small GTPase Rabs. As a Rab3/Rab27 effector protein on secretory vesicles, Rabphilin 3A was implicated to interact with SNAP-25 to regulate vesicle exocytosis in neurons and neuroendocrine cells, yet the underlying mechanism remains unclear. In this study, we have characterized the physiologically relevant binding sites between Rabphilin 3A and SNAP-25. We found that an intramolecular interplay between the N-terminal Rab-binding domain and C-terminal C2AB domain enables Rabphilin 3A to strongly bind the SNAP-25 N-peptide region via its C2B bottom α-helix. Disruption of this interaction significantly impaired docking and fusion of vesicles with the plasma membrane in rat PC12 cells. In addition, we found that this interaction allows Rabphilin 3A to accelerate SNARE complex assembly. Furthermore, we revealed that this interaction accelerates SNARE complex assembly via inducing a conformational switch from random coils to α-helical structure in the SNAP-25 SNARE motif. Altogether, our data suggest that the promotion of SNARE complex assembly by binding the C2B bottom α-helix of Rabphilin 3A to the N-peptide of SNAP-25 underlies a pre-fusion function of Rabphilin 3A in vesicle exocytosis.

The stability of the primed pool of synaptic vesicles and the clamping of spontaneous neurotransmitter release rely on the integrity of the C-terminal half of the SNARE domain of syntaxin-1A

“…Single-molecule optical-tweezer studies have focused on the impact of regulatory molecules on the stability of assembly such as Munc18-1 ( Ma et al, 2015 ; Jiao et al, 2018 ) and complexin ( Hao et al, 2023 ), or on the intrinsic stability of the hydrophobic layers in the step-wise assembly of the SNARE complex ( Gao et al, 2012 ; Ma et al, 2015 ; Zhang, 2017 ). Although the conserved hydrophobic layers in the SNARE domains of STX1A and STX2 ( Figure 1 ) suggest unchanged zippering and intrinsic stability of the complex, further studies addressing the contribution of surface residues on the stability of the alpha-helical structure of the SNARE domain of STX1 ( Li et al, 2022 ) or the stability of the SNARE complex should be conducted.…”