Fusing simulation and experiment: The effect of mutations on the structure and activity of the influenza fusion peptide

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.

Conformational properties, membrane interaction, and antibacterial activity of the peptaibiotic chalciporin A: Multitechnique spectroscopic and biophysical investigations on the natural compound and labeled analogs

“…The observed substantial enhancement is indicative of an effective membrane binding . This finding is not unexpected considering the overall high hydrophobicity of chalciporin A and the extensively reported ability of an exposed Trp residue to act as a hook for a peptide at an aqueous/membrane interface . The binding curve is comparable to those reported previously for fluorescent analogs of trichogin GA IV (although in membranes of different composition) and is consistent with the similar membrane‐perturbing properties reported above.…”

Effect of pH on the influenza fusion peptide properties unveiled by constant-pH molecular dynamics simulations combined with experiment

“…a lipid acyl chain that extends to and beyond the corresponding phosphate group) is a determinant step in membrane fusion 19 . The occurrence of lipid tail protrusion has been observed in several simulation studies of the influenza fusion peptide in membrane bilayers, which indicates that the peptide increases the probability of protrusion events 7,14,18,27 . It has also been shown that the peptide interacts with the lipid headgroups, mainly through the N-terminal group, which induces these headgroups to penetrate deeper into the membrane (headgroup intrusion) 7,14,20,27 .…”

“…The occurrence of lipid tail protrusion has been observed in several simulation studies of the influenza fusion peptide in membrane bilayers, which indicates that the peptide increases the probability of protrusion events 7,14,18,27 . It has also been shown that the peptide interacts with the lipid headgroups, mainly through the N-terminal group, which induces these headgroups to penetrate deeper into the membrane (headgroup intrusion) 7,14,20,27 . A recent study using multiscale simulations indicates that hemagglutinin-catalyzed membrane fusion is a two-stage process: first lipid-tail protrusion induced by the fusion peptide catalyzes stalk formation and second, the fusion peptide and transmembrane domain interact with the distal membrane leaflet leading to hemifusion diaphragm formation and fusion pore opening 27 .…”

“…This region is very conserved across different hemagglutinin subtypes (18 out of 23 residues are strictly conserved among all influenza A strains) and several mutations have been shown to abolish or impair its function 4 . Several in-vitro studies have shown that the isolated fusion peptide promotes lipid mixing of large unilamellar vesicles, which evidences that the peptide induces hemifusion, even in the absence of the rest of hemagglutinin [5][6][7][8] . The peptide structure in detergent micelles has been analyzed by NMR studies; it has a helix-turn-helix structure in which the angle between the helices depends on the peptide length [9][10][11][12] .…”

Exploring high‐dimensional free energy landscapes of chemical reactions

“…Literature of BEMetaD also includes conformational sampling in α ‐helical glycoproteins, Bovine Chymosin, peptides, conformational sampling of intrinsically disordered as well as other proteins, conduction through ion channels, and protein aggregation . The method has also helped to resolve the structure of large and complex systems such as protein–RNA complex, and membrane inserted influenza fusion peptide . Clearly, a substantial number of complex biophysical problems have been addressed using this approach.…”