Role of the transmembrane domain in SNARE protein mediated membrane fusion: peptide nucleic acid/peptide model systems

Abstract

Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented.

The Multifaceted Role of SNARE Proteins in Membrane Fusion

“…The transition from hemifusion to fusion was reported to be blocked in SNARE-mediated proteoliposome fusion when the C-terminal half of SNARE TMD was deleted (Xu et al, 2005). Similarly, a series of truncated mutants reported for the sybII TMD was largely incompetent to support neurosecretion in PC12 cells (Fdez et al, 2010) and the lipid mixing was significantly reduced and fusion completely diminished in SNARE-mimics upon TMD shortening (Wehland et al, 2016), suggesting a stringent length requirement for the transmembrane domain.…”

“…The formation of the fusion pore was suggested to be initiated by the movement of the sybII TMD uncharged C-terminus into the membrane interior, induced by the pulling force resulting from SNARE complex zippering, as revealed by coarse-grained simulations (Lindau et al, 2012) (see Figure 5). Addition of charged residues to the C-terminus of SNARE TMD inhibited exocytosis in chromaffin and PC 12 cells (Ngatchou et al, 2010; Wehland et al, 2016), suggesting a mechanism in which the movement of the C-terminus initiates the fusion pore formation by rearranging the bilayer structure in distal leaflets (Fang and Lindau, 2014). …”

Interaction of SNARE Mimetic Peptides with Lipid bilayers: Effects of Secondary Structure, Bilayer Composition and Lipid Anchoring

“…The proposed working mechanism of these protein complexes is the zipper-like assembly of a coiled-coil bundle of four alpha helices, which assists membrane docking and overcomes the water barrier between the two membranes 1,2 . The SNARE protein machinery has inspired the design of simplified model fusion systems based on lipid vesicles, which aim to mimic the zipper like folding mechanism using DNA 3–7 , PNA 8–10 , peptides 11–15 or other molecules 16 . The complementary coiled-coil forming peptides shown in Fig.…”

Extending the x-ray study of membrane fusion in supported multibilayers towards physiological conditions

“…Starting from such a success, our collaborators [104] introduced recognition units (β-glutamine) into the current peptide sequence, enabling helix-helix assembly in a sequence-controlled manner. In the future, these results can be further utilized to design and synthesize the TMDs of SNARE analogs, in association with various fusion recognition motifs [105][106][107] and linkers [108,109].…”