Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Funder: National Collaborative Research Infrastructure Strategy (NCRIS)Funder: ANR/NSF-PIREFunder: Science and Technology Facilities CouncilFunder: Institut Laue LangevinCoronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Funder: National Collaborative Research Infrastructure Strategy (NCRIS)Funder: ANR/NSF-PIREFunder: Science and Technology Facilities CouncilFunder: Institut Laue LangevinCoronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed

Developing advanced models of biological membranes with hydrogenous and deuterated natural glycerophospholipid mixtures

Cellular membranes are complex systems that consist of hundreds of different lipid species. Their investigation often relies on simple bilayer models including few synthetic lipid species. Glycerophospholipids (GPLs) extracted from cells are a valuable resource to produce advanced models of biological membranes. Here, we present the optimisation of a method previously reported by our team for the extraction and purification of various GPL mixtures from Pichia pastoris. The implementation of an additional purification step by High Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD) enabled for a better separation of the GPL mixtures from the neutral lipid fraction that includes sterols, and also allowed for the GPLs to be purified according to their different polar headgroups. Pure GPL mixtures at significantly high yields were produced through this approach. For this study, we utilised phoshatidylcholine (PC), phosphatidylserine (PS) and phosphatidylglycerol (PG) mixtures. These exhibit a single composition of the polar head, i.e., PC, PS or PG, but contain several molecular species consisting of acyl chains of varying length and unsaturation, which were determined by Gas Chromatography (GC). The lipid mixtures were produced both in their hydrogenous (H) and deuterated (D) versions and were used to form lipid bilayers both on solid substrates and as vesicles in solution. The supported lipid bilayers were characterised by quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR), whereas the vesicles by small angle X-ray (SAXS) and neutron scattering (SANS). Our results show that despite differences in the acyl chain composition, the hydrogenous and deuterated extracts produced bilayers with very comparable structures, which makes them valuable to design experiments involving selective deuteration with techniques such as NMR, neutron scattering or infrared spectroscopy.We are grateful to the ILL and the ESRF for awarding beamtimes (DOI: 105291/ILL-DATA.EASY-975) and (DOI: https://doi.org/10.15151/ESRF-DC-1026409781) respectively. Lipids were produced in the L-Lab (www.ill.eu/L-Lab) facility within the PSCM initiative at the ILL from biomass prepared in the D-Lab. We are grateful to Hanna Wacklin-Knecht (ESS) for useful discussions. This project received funding from the European Union's Horizon 2020 research and innovation program under grant agreement N 654000 (SINE2020) and from the League of advanced European Neutron Sources (LENS). CB, YYB and the GEMELI Lipidomic platform were supported by Agence Nationale de la Recherche, France (Project ApicoLipiAdapt grant ANR-21-CE44-0010), the Fondation pour la Recherche Médicale (FRM EQU202103012700), Laboratoire d'Excellence Parafrap, France (grant ANR-11-LABX-0024), LIA-IRP CNRS Program (Apicolipid project), the Université Grenoble Alpes (IDEX ISP Apicolipid), Indo-French Collaborative Research Program Grant CEFIPRA (Project 6003-1), and Région Auvergne Rhone-Alpes for the lipidomics analyses platform (Grant IRICE Project GEMELI). A.M. acknowledges the financial support from MICINN under grant PID2021-129054NA-I00.Peer reviewe

Investigation on the relationship between lipid composition and structure in model membranes composed of extracted natural phospholipids

Unravelling the structural diversity of cellular membranes is a paramount challenge in life sciences. In particular, lipid composition affects the membrane collective behaviour, and its interactions with other biological molecules. Here, the relationship between membrane composition and resultant structural features was investigated by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry on in vitro membrane models of the mammalian plasma and endoplasmic-reticulum-Golgi intermediate compartment membranes in the form of Langmuir monolayers. Natural extracted yeast lipids were used because, unlike synthetic lipids, the acyl chain saturation pattern of yeast and mammalian lipids are similar. The structure of the model membranes, orthogonal to the plane of the membrane, as well as their lateral packing, was found to depend strongly on their specific composition, with cholesterol having a major influence on the in-plane morphology, yielding a coexistence of liquid-order and liquid-disorder phases

Investigation on the relationship between lipid composition and structure in model membranes composed of extracted natural phospholipids

[Hypothesis] Unravelling the structural diversity of cellular membranes is a paramount challenge in life sciences. In particular, lipid composition affects the membrane collective behaviour, and its interactions with other biological molecules.[Experiments] Here, the relationship between membrane composition and resultant structural features was investigated by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry on in vitro membrane models of the mammalian plasma and endoplasmic-reticulum-Golgi intermediate compartment membranes in the form of Langmuir monolayers. Natural extracted yeast lipids were used because, unlike synthetic lipids, the acyl chain saturation pattern of yeast and mammalian lipids are similar.[Findings] The structure of the model membranes, orthogonal to the plane of the membrane, as well as their lateral packing, were found to depend strongly on their specific composition, with cholesterol having a major influence on the in-plane morphology, yielding a coexistence of liquid-order and liquid-disorder phases.The authors thank the Institut Laue-Langevin (DOI: 10.5291/ILL-DATA.DIR-215) for financial support and allocation of beamtime, the ILL D-Lab for provision of the hydrogenous and deuterated yeast cells and the Partnership for Soft Condensed Matter (PSCM) for the lab support and provision of lipid extracts (L-Lab). Part of the lipid extraction activity was funded by the ANR/NSF-PIRE project REACT (Research and Education in Active Coatings Technologies for Human Health) and the Ligue for Advanced Neutron Sources (LENS). E.G. and A.M. received financial support from MICINN (grants PID2019-106557GB-C21 and PID2021-129054NA-I00, respectively), N.R.Z. from the Wellcome Trust (grant 207455/Z/17/Z). A. M. also acknowledges the financial support received from the IKUR Strategy under the collaboration agreement between Ikerbasque Foundation and Materials Physics Center on behalf of the Department of Education of the Basque Government. The National Deuteration Facility in Australia is partly funded by The National Collaborative Research Infrastructure Strategy (NCRIS), an Australian Government initiative.Peer reviewe