SNARE Modulators and SNARE Mimetic Peptides

Abstract

The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins play a central role in most forms of intracellular membrane trafficking, a key process that allows for membrane and biocargo shuffling between multiple compartments within the cell and extracellular environment. The structural organization of SNARE proteins is relatively simple, with several intrinsically disordered and folded elements (e.g., SNARE motif, N-terminal domain, transmembrane region) that interact with other SNAREs, SNARE-regulating proteins and biological membranes. In this review, we discuss recent advances in the development of functional peptides that can modify SNARE-binding interfaces and modulate SNARE function. The ability of the relatively short SNARE motif to assemble spontaneously into stable coiled coil tetrahelical bundles has inspired the development of reduced SNARE-mimetic systems that use peptides for biological membrane fusion and for making large supramolecular protein complexes. We evaluate two such systems, based on peptide-nucleic acids (PNAs) and coiled coil peptides. We also review how the self-assembly of SNARE motifs can be exploited to drive on-demand assembly of complex re-engineered polypeptides.

Keywords: SNARE mimetic; SNARE motif; SNARE peptide; SNARE protein; SNAREpins; clostridial neurotoxins; functional peptide; fusogen; membrane fusion; molecular self-assembly.

Figure 1

Molecular anatomy of SNARE proteins and their complexes. (a) Model of rat VAMP2 inserted into lipid bilayer. Amino acid residues indicate boundaries of VAMP2 structural regions (TMR denotes transmembrane region). PDB accession#: 2KOG. (b) Structure of an N-terminal peptide of yeast SNARE protein Sed5p (red) in a complex with yeast SM protein Sly1p (blue). PDB accession#: 1MQS. (c) Structure of the rat neuronal SNARE complex. PDB accession#: 3HD7.

Figure 2

Mapping of the known SNARE-derived functional peptides. Neuronal SNARE proteins, (a) SNAP-25, (b) VAMP2, and (c) syntaxin 1A are schematically shown with N-terminal domain, SNARE motifs and transmembrane region (TMR) indicated. Numbering of amino acid residues is shown below in black for rat SNARE proteins. Functional peptides are mapped by red bars. The numbering (in red) corresponds to the peptide entry number in the Supplementary Table S1.

Figure 3

SNARE mimicry by peptide-nucleic acid (PNA) fusogens. PNA pairs were inserted into artificial liposomes via native transmembrane regions of SNARE proteins (modified from [108]). SUV—small unilamellar vesicle. Optimal length of the PNA linker for membrane fusion is shown in angstroms. Tm of DNA duplex is indicated. Antiparallel orientation is shown.

Figure 4

SNARE mimicry by E/K coiled coil peptides. E (blue coil) and K (red coil) peptides inserted into artificial membranes by a covalently linked lipid moiety. One peptide is positively charged (+) and additionally interacts with membrane, another peptide is negatively charged (-). Antiparallel orientation is shown. GUV—giant unilamellar vesicle. SUV—small unilamellar vesicle. Each peptide is lipidated (in grey: Cho—cholesterol, DOPE—1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine) and contains a PEG linker of variable length (in green). Sequence of a basic (in red) and an acidic (in blue) heptad repeat is shown. Three or four repeats are included for optimal performance.

Figure 5

SNARE tagging. (a) On-demand linking of surface-derivatised quantum dots to the receptor-binding domain of botulinum neurotoxin (Rbd) using the SNARE domain of a native SNAP-25 protein as a ‘staple’. (b) Self-assembly linking of SNARE-tagged botulinum neurotoxin domains into a functional toxin using SNARE domain of native syntaxin protein as a ‘staple’; (a) and (b) reproduced with permission from [133]. (c) Stepwise assembly of an artificial clostridial chimeric protein built from botulinum type A protease/translocation domain from Clostridium botulinum (devoid of motor-neuron-targeting domain) and the tetanus toxin binding domain from Clostridium tetani (capable of targeting central neurons, but not motoneurons). A SNARE domain of native syntaxin protein is used as a ‘staple’. Reproduced under CC-BY-4.0 license from [134].

Figure 6

SNARE-inspired ‘stapling’ of proteins together and the effect of the ‘staple’ peptide length on the stability of the assembled complex. Both linkers and the ‘staple’ are based on native SNARE motif polypeptides. The ‘staple’ is a synthetic peptide based on syntaxin sequence. (a) The overall scheme explaining the experiment where two large proteins were connected using a short synthetic peptide. The fully assembled complex is stable in 1% SDS solution. (b) Stability of stapling by the indicated peptides under denaturing conditions. Bars indicate the amount in resonance units (measured with SPR Biacore technology) of linker B remaining after exposure of the complex to 1% SDS. (c) Different length ‘staple’ peptides used; all sequences are based on the SNARE motif of native syntaxin protein. Reproduced from [137] under the ACS AuthorChoice/Editors’ Choice usage agreement.

Figure 7

SNARE-inspired re-engineered binary PPI interface for on-demand assembly and controlled disassembly of proteins. (a) Schematic of the native SNARE proteins and their transmembrane topology. Color code: domain V from VAMP2 (synaptobrevin protein) is shown in blue, domain S from syntaxin is shown in red, linker and the two SNARE domains from SNAP-25 are shown in green. Cylinders represent α-helixes; the yellow rectangle represents the membrane into which the native SNARE proteins are embedded via an α-helical transmembrane domain (VAMP2 and syntaxin) or palmitoylated cysteine residues (4 zig-zag segments on the SNAP-25). (b) Schematic of the re-engineered SNARE mimetics. Cylinders represent SNARE domains highlighted in panel A, whereas the green lines correspond to the naturally occurring linker between the two α-helices of SNAP-25. (c) Stability and temperature-dependent disassembly of the SNARE-inspired re-engineered binary PPI complex measured using far-UV synchrotron radiation circular dichroism (SRCD). The range of temperature in which control of protein disassembly is possible extends from Tm = 43 °C to Tm = 80 °C. All panels are reproduced with permission from [138].