The SNAP-25 linker supports fusion intermediates by local lipid interactions

Abstract

SNAP-25 is an essential component of SNARE complexes driving fast Ca2+-dependent exocytosis. Yet, the functional implications of the tandem-like structure of SNAP-25 are unclear. Here, we have investigated the mechanistic role of the acylated “linker” domain that concatenates the two SNARE motifs within SNAP-25. Refuting older concepts of an inert connector, our detailed structure-function analysis in murine chromaffin cells demonstrates that linker motifs play a crucial role in vesicle priming, triggering, and fusion pore expansion. Mechanistically, we identify two synergistic functions of the SNAP-25 linker: First, linker motifs support t-SNARE interactions and accelerate ternary complex assembly. Second, the acylated N-terminal linker segment engages in local lipid interactions that facilitate fusion triggering and pore evolution, putatively establishing a favorable membrane configuration by shielding phospholipid headgroups and affecting curvature. Hence, the linker is a functional part of the fusion complex that promotes secretion by SNARE interactions as well as concerted lipid interplay.

Roles for the SNAP25 Linker Domain in the Fusion Pore and a Dynamic Plasma Membrane SNARE Acceptor Complex

“…The domain in SNAP25 that links the two SNARE motifs can no longer be considered a minor player in exocytosis as had been suggested by earlier work (Chen et al, 1999). The linker participates in the formation of the t-SNARE receptor complex with syntaxin (Jiang et al, 2019;Shaaban et al, 2019), is required for robust secretion (Shaaban et al, 2019;Wang et al, 2008), and functions to quicken fusion pore expansion and to control secretion rates (Shaaban et al, 2019). Normal function requires precise localization of the palmitoylated residues in the linker.…”

“…The two motifs, SN1 and SN2, are joined by a linker sequence (light blue and yellow line) with four N-terminal cysteines that are sites for palmitoylation. The linker region is divided into N-terminal (blue) and C-terminal (yellow) domains according to the constructs in Shaaban et al (Shaaban et al, 2019). SN2 contains the cleavage site for botulinum neurotoxin type E (BoNT E).…”

“…Botulinum neurotoxin type E (BoNT E) was used in an earlier study to inactivate endogenous SNAP25 (Wang et al, 2008). Shaaban et al (Shaaban et al, 2019) demonstrate that the N-and Cterminals domains have different roles in enabling the function of SNAP25 in exocytosis. The horizontal distances are to scale.…”

“…The Cterminal linker segment ( Fig. 1, yellow line) strongly facilitates the in vitro binding of SN2 to the readily formed complexes of SN1 and syntaxin (Shaaban et al, 2019). It was independently found that the linker-membrane interaction increases linker helical content, which enables the C-terminal linker region to interact with syntaxin (Jiang et al, 2019).…”

The Linker Domain of SNAP25 Acts as a Flexible Molecular Spacer to Ensure Efficient S-Acylation

“…In contrast, the sequence of the mini-linker is less important for efficient Sacylation as long as it confers structural flexibility. Future work will determine if the sequence of the mini-linker is important for the function of SNAP25 in fusion pore dynamics and exocytosis [33].…”