Abstract
During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide’s free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies.
Highlights
The fusion peptide comprises the first ~23 amino acid residues of HA2 and this segment is able to induce lipid mixing of liposomes, even in the absence of the rest of the protein[8,9]
Upon exposure to the low pH of late endosomes, the fusion peptide (FP) is extruded from this pocket and becomes exposed to water, before it inserts into the host membrane
Our results indicate that the G1V, W14A and G12A/G13A mutants had a similar effect to the WT peptide on the order of the surrounding lipids, whereas the G4A/G8A/G16A/G20A mutant had a stronger influence on the order parameters of the acyl chain 2 (Fig. 7)
Summary
The fusion peptide comprises the first ~23 amino acid residues of HA2 and this segment is able to induce lipid mixing of liposomes (hemifusion), even in the absence of the rest of the protein[8,9]. The substitution of G8 (which is part of the glycine zipper) by an alanine resulted in the loss of activity[19], and the NMR structure of this mutant in DPC micelles revealed that it tends to adopt an open conformation[15] This supports the notion that the close contact between glycine pairs located on the two helices (glycine zipper) is important to maintain the helical-hairpin structure, which seems to be determinant for the peptide’s function. Atomistic self-assembly simulations performed by our group, in which the membrane spontaneously grows around the peptide, avoiding the bias imposed by choosing the initial peptide location[2], revealed that the peptide can adopt two different configurations: parallel to the membrane in the headgroup-lipid tail interface or a considerably more tilted membrane-spanning conformation. When the peptide is in the tilted conformation it has a stronger effect on the membrane, lowering the bilayer thickness, disordering nearby lipids, and promoting lipid tail protrusion
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