Coiled-coil driven membrane fusion: zipper-like vs. non-zipper-like peptide orientation

Abstract

Membrane fusion plays a central role in biological processes such as neurotransmission and exocytosis. An important class of proteins that induce membrane fusion are called SNARE (soluble N-ethyl malemeide sensitive factor attachment protein receptors) proteins. To induce membrane fusion, two SNARE proteins embedded in opposing membranes form a four-helix coiled-coil motif together with a third, cytoplasmic, SNARE protein. Coiled-coil formation brings the two membranes into close proximity allowing fusion to occur. Importantly, structural investigations have demonstrated that native membrane fusion only occurs when the orientation of the coiled-coil motif resembles that of a zipper. The zipper orientation arises when parallel coiled-coil formation takes place between peptides that are anchored into apposing membranes at identical termini, thereby forcing the membranes into close contact. Recently, we have designed a synthetic model for membrane fusion, which is based on a set of lipidated coiled-coil forming peptide pairs which are denoted E-K. When incorporated into liposomal membranes, coiled-coil formation between these lipidated peptides induces targeted and efficient membrane fusion of liposomes. Our model system mimics SNARE-driven membrane fusion, as it contains a coiled-coil motif which has a zipper-like orientation, similar to that of the SNARE proteins. Here we investigate whether the zipper-like orientation of the coiled-coil motifs is a prerequisite for membrane fusion in our model system. Our strategy is based on conjugation of the transmembrane anchor to either the N- or the C-terminus of peptides E and K. Whereas the use of a set of complementary peptides with the membrane anchor on identical peptide termini yields the zipper-like orientation of the coiled-coil complex, membrane anchors on opposite peptide termini results in a non-zipper-like coiled-coil orientation. Surprisingly, it was observed that efficient and targeted membrane fusion was induced even when the coiled-coil motif did not form the zipper-like orientation. This demonstrates that for our model system, the zipper model for membrane fusion does not apply.

A Coiled-Coil Peptide Shaping Lipid Bilayers upon Fusion

“…Fig. 2 A displays the change of fluorescence intensity upon varying the lipid to peptide ratio by addition of vesicles of the composition DOPC:DOPE:cholesterol (molar ratio: 2:1:1), as used in recent vesicle fusion studies (5)(6)(7)(8)(9)(10)16,44,45). For comparative purposes, the total concentration of the tryptophan bearing peptide (X*, X ¼ K or E, respectively) was kept constant.…”

“…Therefore for further quenching measurements at constant lipid concentrations, this ratio was chosen. For measurements with LPE* and LPK*, a ratio of 100:1 was chosen because this reflects the conditions of typical vesicle fusion experiments (5)(6)(7)(8)(9)(10)16,44,45).…”

Interplay between Lipid Interaction and Homo-coiling of Membrane-Tethered Coiled-Coil Peptides

“…[1][2][3] Amongst those, membrane tethered coiled-coil forming lipopeptides whose design has been inspired by natural SNARE (soluble NSF attachment protein receptor) proteins have been examined extensively in different studies because they were shown to trigger specific and leakage free full fusion of vesicles. 1,[4][5][6][7][8][9][10][11][12][13][14][15] The lipopeptides used here consist of a cholesterol lipid anchor connected via a polyethylene glycol (PEG 12 ) spacer to the peptide moieties (lipopeptides CPE, CPK; Chart 1A). The peptide recognition unit is made of two hetero coiled-coil forming peptides called E: (EIAALEK) 3 -NH 2 and K: (KIAALKE) 3 -NH 2 .…”

A non-zipper-like tetrameric coiled coil promotes membrane fusion

“…CC-K/E is known to induce efficient and targeted membrane fusion when both peptides are modied with a lipid anchor and incorporated into liposome membranes. 20,22,23,26 These fusogenic constructs comprise three distinct segments: the peptide segment, which serves as the recognition unit; a hydrophobic cholesterol anchor, which enables the construct to be secured in a liposome membrane, and; a hydrophilic linker, which is thought to be crucial for allowing force transduction and therefore helping to facilitate fusion in natural SNARE systems. 6,50,51 In addition to the linking region being crucial for fusion, it is postulated that the peptides need to be in a parallel orientation, as their 'zippering' from one terminus to the other as they fold provides the force that draws the two membranes close together and induces them to fuse.…”

“…[20][21][22][23] It has been postulated that, for fusion to occur, the fusogens need to be aligned in a parallel orientation allowing them to 'zipper' up from their distal N-termini to their membraneproximal C-termini, generating an inward force that pulls the two membranes together and forces them to fuse. 24,25 To probe this 'zipper' hypothesis, peptides that are anchored into the membrane at opposing termini, 26 or peptides that have an antiparallel orientation can be used, Scheme 1. Previous studies have shown that both antiparallel SNARE derivatives and short antiparallel coiled coils cannot induce fusion.…”