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

Jan-Dirk Wehland,Ulf Diederichsen,Samit Guha,Pawan Kumar,Barbara E Hubrich,Reinhard Jahn,Antonina S Lygina

doi:10.1039/c6mb00294c

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.

Highlights

Membrane fusion is an indispensable event in all forms of life
The design of SNARE protein mimics is based on the transmembrane domains (TMDs) and linker regions of native syntaxin-1A and synaptobrevin 2, whereas a peptide nucleic acids (PNAs)–PNA double strand replaces the tetrameric SNARE motif
The N-terminal ends of TMD/linker domains of native synaptobrevin 2 and syntaxin-1A were extended with PNA1 and PNA2, respectively

Summary

Introduction

Membrane fusion is an indispensable event in all forms of life. Intracellular fusion is mediated by specific molecular recognition of SNARE (Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor) proteins. Most of these proteins contain a C-terminal transmembrane domain (TMD) connected by a short linker to the recognition sequence assembling in the latter SNARE complex. Fusion of two membranes is facilitated by the interaction of the key SNARE proteins syntaxin-1A and SNAP-25 residing in the plasma membrane and synaptobrevin 2 in the vesicle membrane.[1,2] Several mechanisms of SNARE

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Results

Conclusion