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

Abstract

The designed coiled-coil-forming peptides E [(EIAALEK)3] and K [(KIAALKE)3] are known to trigger efficient membrane fusion when they are tethered to lipid vesicles in the form of lipopeptides. Knowledge of their secondary structure is a key element in understanding their role in membrane fusion. Special conditions can be found at the interface of the membrane, where the peptides are confined in close proximity to other peptide molecules as well as to the lipid interface. Consequently, different structural states were proposed for the peptides when tethered to this interface. Due to the multitude of possible states, determining the structure solely on the basis of circular dichroism (CD) spectra at a single temperature can be misleading. In addition, it has not yet been possible to unambiguously distinguish between the membrane-bound and the coiled-coil states of these peptides by means of infrared (IR) spectroscopy due to their very similar amide I' bands. Here, the molecular basis of this similarity is investigated by means of site-specific (13)C-labeled FTIR spectroscopy. Structural similarities between the membrane-interacting helix of K and the homo-coiled-coil-forming helix of E are shown to cause the similar spectroscopic properties. Furthermore, the peptide structure is investigated using temperature-dependent CD and IR spectroscopy, and it is shown that the different states can be distinguished on the basis of their thermal behavior. It is shown that the two peptides behave fundamentaly differently when tethered to the lipid membrane, which implies that their role during membrane fusion is different and the mechanism of this process is asymmetric.

Geometry of the Contact Zone between Fused Membrane-Coated Beads Mimicking Cell-Cell Fusion

“…Furthermore, interaction of the K-peptide with the lipid membrane could influence lipid composition and local curvature, facilitating membrane reorganization within the contact zone. Enrichment of cholesterol in the vicinity of K-peptide membrane interaction has been suggested (23,24). Thus, energy considerations suggest a minimal geometry, consistent with the finding that a minimal angle is needed to account for large diffusion slowdowns compared to the reference experiment on single spheres.…”

A Coiled-Coil Peptide Shaping Lipid Bilayers upon Fusion

“…In general, the E* and LPE* are therefore not embedded in the membrane. Whereas E* showed no increased helicity, the enhanced helicity of LPE* in these systems can be attributed to the known tendency of E* to form homodimers at increased local concentration (20).…”

“…Recent infrared spectroscopic studies of LPE and LPK containing lipid mono-or bilayers revealed differing behaviors of the two peptides before the fusion commences (19,20). The negatively charged E shows relatively weak interactions with the lipid interface, staying in the water phase in the form of homomeric coiled-coil dimers E/E.…”

“…However, it has not yet been proven that heterodimeric coiled-coil formation and K membrane interaction coincide on the membrane interface, because the peptide state could only be determined unambiguously in systems where only E or only K were present. The increase in helicity that accompanies both coiled-coil formation and membrane incorporation hampered an unequivocal interpretation of infrared and circular dichroism spectra in vesicle systems with both peptides present (20). Thus, it remains unclear if one peptide state-membrane-bound or coiled-coil-is predominant on the membrane during the course of fusion and afterwards.…”

“…It was hypothesized that the direct K-membrane interactions lead to distortion of the bilayers or increased local curvature and thereby promote the merging of lipid bilayers (19,20). In this model the two peptides E and K have different functions apart from coiled-coil formation, leaving multiple tasks to K. However, very little is known about the structural consequences of the insertion of the amphipathic helix of K into lipid membranes.…”