Lipid Bilayer Degradation Induced by SARS-CoV-2 Spike Protein as Revealed by Neutron Reflectometry

Abstract

SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes, formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements show that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.

Synthesis of Perdeuterated Linoleic Acid‐d31 and Chain Deuterated 1‐Palmitoyl‐2‐linoleoyl‐sn‐glycero‐3‐phosphocholine‐d62

“…[40] After 30 hours, a small amount (5%) of the migrated product was observed. In a similar manner, Roberts and co-workers did not observe a measurable amount of OPPC by NMR measurements after the Steglich esterification of 1-palmitoyl-2-hydroxy-sn-glycero-3phosphocholine using 13 C labelled oleic acid. [41] Standard conditions for the esterification of 1palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine generally call for an excess of the carboxylic acid to ensure sufficient conversion of the poorly nucleophilic secondary hydroxyl group to the desired ester.…”

“…Rather, a more traditional approach was taken, starting from deuterated pentanoic acid 9 (Scheme 3). The acid 9 was reduced with lithium aluminium deuteride and immediately activated as the tosylate (13). Protected propargyl alcohol 14 was synthesised in a three-step procedure reported by Dodo and co-workers.…”

“…Differentiation and quantification of regioisomeric diacylglycerol phospholipids is immensely challenging. For instance, comparison of the NMR spectra of commercially sourced POPC and OPPC shows no difference in the 1 H, 31 P NMR spectra and only marginal differences in the 13 C spectra. The two carbonyl carbons of POPC and OPPC are separable, and can be used to estimate the ratio of the two species.…”

“…The two carbonyl carbons of POPC and OPPC are separable, and can be used to estimate the ratio of the two species. The carbonyl region of the 13 C NMR spectrum of 20 indicated the formation of one predominant regioisomer. However, due to the low abundance of the 13 C isotope, accurate quantification is not possible.…”

“…[12] Of note, the use of deuterated lipids has been instrumental in determining how SARS-CoV-2 interacts with cell membranes. [13] In addition to neutron experiments, [14] deuterated phospholipids are required for particular NMR [15] and vibrational spectroscopy studies. [16] Monoglyceridebased liquid crystals have been increasingly used as extended-release drug delivery systems using glyceryl monooleate (GMO) and monolinoleate (GML).…”