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

www.nature.com/scientificreports OPEN Effect of pH on the influenza fusion peptide properties unveiled by constant‑pH molecular dynamics simulations combined with experiment Diana Lousa1*, Antónia R. T. Pinto2, Sara R. R. Campos1, António M. Baptista1, Ana S. Veiga2, Miguel A. R. B. Castanho2 & Cláudio M. Soares1* The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment. Acidification triggers a large conformational change in the fusion protein, hemagglutinin (HA), which enables the insertion of the N-terminal region of the HA2 subunit, known as the fusion peptide, into the membrane of the host endosome. However, the mechanism by which pH modulates the molecular properties of the fusion peptide remains unclear. To answer this question, we performed the first constant-pH molecular dynamics simulations of the influenza fusion peptide in a membrane, extending for 40 µs of aggregated time. The simulations were combined with spectroscopic data, which showed that the peptide is twofold more active in promoting lipid mixing of model membranes at pH 5 than at pH 7.4. The realistic treatment of protonation introduced by the constant-pH molecular dynamics simulations revealed that low pH stabilizes a vertical membrane-spanning conformation and leads to more frequent contacts between the fusion peptide and the lipid headgroups, which may explain the increase in activity. The study also revealed that the N-terminal region is determinant for the peptide’s effect on the membrane. Influenza infections affect a very large number of individuals every year and represent a serious social and economic burden1. The situation becomes even more dramatic when a new pandemic arises, which can lead to very high mortality rates1. Currently there is no universal and effective therapy against this virus and, thus, it is crucial to obtain a detailed understanding of the infectious process and its key players. One important step of this process is the fusion between the viral and host membranes, catalyzed by the fusion protein hemagglutinin, which is one of the most promising drug targets against this virus2. Hemagglutinin is a homotrimer and each monomer is composed of two polypeptide chains, named HA1 and HA2, connected by a disulfide bond. HA1 is responsible for binding to the sialic acid receptors on the host membrane, whereas HA2 contains the fusion machinery3. After binding to the receptors on the host cell, the influenza virus is uptaken by endocytosis. Release of the genetic material of the virus into the host cell occurs at the endosome membrane level. At the late endosomes there is a pH drop to a value of around 5 that triggers a large conformational change of HA, which is crucial for the fusion process3. The first 23 amino acid residues of HA2 are particularly important in the fusion process, since this region inserts into the host membrane, promoting fusion and is, therefore, known as the fusion peptide4. 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 f unction4. 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 h emagglutinin5–8. 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 length9–12. A 20-residue long fusion peptide (HAfp-20) 1 ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780‑157 Oeiras, Portugal. 2Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Professor Egas Moniz, 1649‑028 Lisboa, Portugal. *email: ; Scientific Reports | (2020) 10:20082 | https://doi.org/10.1038/s41598-020-77040-y 1 Vol.:(0123456789) www.nature.com/scientificreports/ adopts an open inverted-V structure at pH 5 9, whereas a 23-residue long peptide (HAfp-23) displays a more closed helical-hairpin structure, both at pH 4 and 7.410. A crucial issue that remains unclear is the orientation adopted by the fusion peptide in the membrane bilayer, as it is determinant for peptide-induced perturbation of lipid b ilayers13. Based on the NOE interactions between the amide protons of the HAfp-23 and dodecylphosphocholine micelle protons, Lorieau et al. concluded that one of the peptide sides is exposed to water and postulated that the peptide adopts an interfacial conformation10. However, since their results were obtained in a micelle environment, which has a very different structure from a membrane bilayer, having a single lipidic layer and a considerably higher curvature, it is not clear if the peptide adopts this type of arrangement in the membrane. Molecular dynamics (MD) simulations performed by us, indicated that the peptide can adopt two different conformations in a membrane bilayer: an interfacial orientation, parallel to the membrane surface and a membrane-spanning orientation, perpendicular to the b ilayer14. Subsequent simulation studies by Worch et al. using temperature replica exchange molecular dynamics also found that the influenza fusion peptide can adopt these two configurations and indicate that he membranespanning configuration corresponds to the lowest free energy minimum for the 23-residue long fusion peptide with a charged N-terminus15,16. The mechanism by which the peptide induces lipid mixing is not fully elucidated, but several recent experimental and computational studies have suggested different modes of action, including altering the membrane curvature, increasing or decreasing lipid order or inducing pore formation and s tabilization14–26. A mechanism that has been proposed based on molecular dynamics (MD) simulations asserts that lipid tail protrusion (i.e. a lipid acyl chain that extends to and beyond the corresponding phosphate group) is a determinant step in membrane fusion19. 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 e vents7,14,18,27. 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 dista (...truncated)