tetano
Editor, Senior Moderator
Biophys J
. 2021 Dec 17;S0006-3495(21)03948-5.
doi: 10.1016/j.bpj.2021.12.024. Online ahead of print.
Importance of Negatively Charged Residues in the Membrane Ordering Activity of SARS-CoV-1 and -2 Fusion Peptides
Alex L Lai[SUP] 1 [/SUP], Jack H Freed[SUP] 1 [/SUP]
Affiliations
Abstract
Entry of coronaviruses into host cells is mediated by the viral spike (S) protein. Previously, we identified the bona fide FPs for SARS-CoV ("SARS-1") and SARS-CoV-2 ("SARS-2") using ESR spectroscopy. We also found that their FPs induce membrane ordering in a Ca[SUP]2+[/SUP]-dependent fashion. Here we study which negatively charged residues in SARS-1 FP are involved in this binding, to build a topological model and clarify the role of Ca[SUP]2+[/SUP]. Our systematic mutation study on the SARS-1 FP shows that all six negatively charged residues contribute to the FP's membrane ordering activity, with D812 the dominant residue. The corresponding SARS-2 residue D830 plays an equivalent role. We provide a topological model of how the FP binds Ca[SUP]2+[/SUP] ions: its two segments FP1 and FP2 each bind one Ca[SUP]2+[/SUP]. The binding of Ca[SUP]2+[/SUP], the folding of FP (both studied by ITC experiments), and the ordering activity correlate very well across the mutants, suggesting that the Ca[SUP]2+[/SUP] helps the folding of FP in membranes to enhance the ordering activity. Using a novel pseudotyped virus particle (PP)-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole S protein in its trimer form in real time. We found that the SARS-1 and SARS-2 PPs also induce membrane ordering to the extent that separate FPs do, and mutations of the negatively charged residues also significantly suppress the membrane ordering activity. However, the slower kinetics of the FP ordering activity vs. that of the PP suggests the need for initial trimerization of the FPs.
Keywords: Membrane fusion; coronavirus; electron spin resonance; spike glycoprotein; viral entry.
. 2021 Dec 17;S0006-3495(21)03948-5.
doi: 10.1016/j.bpj.2021.12.024. Online ahead of print.
Importance of Negatively Charged Residues in the Membrane Ordering Activity of SARS-CoV-1 and -2 Fusion Peptides
Alex L Lai[SUP] 1 [/SUP], Jack H Freed[SUP] 1 [/SUP]
Affiliations
- PMID: 34929193
- DOI: 10.1016/j.bpj.2021.12.024
Abstract
Entry of coronaviruses into host cells is mediated by the viral spike (S) protein. Previously, we identified the bona fide FPs for SARS-CoV ("SARS-1") and SARS-CoV-2 ("SARS-2") using ESR spectroscopy. We also found that their FPs induce membrane ordering in a Ca[SUP]2+[/SUP]-dependent fashion. Here we study which negatively charged residues in SARS-1 FP are involved in this binding, to build a topological model and clarify the role of Ca[SUP]2+[/SUP]. Our systematic mutation study on the SARS-1 FP shows that all six negatively charged residues contribute to the FP's membrane ordering activity, with D812 the dominant residue. The corresponding SARS-2 residue D830 plays an equivalent role. We provide a topological model of how the FP binds Ca[SUP]2+[/SUP] ions: its two segments FP1 and FP2 each bind one Ca[SUP]2+[/SUP]. The binding of Ca[SUP]2+[/SUP], the folding of FP (both studied by ITC experiments), and the ordering activity correlate very well across the mutants, suggesting that the Ca[SUP]2+[/SUP] helps the folding of FP in membranes to enhance the ordering activity. Using a novel pseudotyped virus particle (PP)-liposome methodology, we monitored the membrane ordering induced by the FPs in the whole S protein in its trimer form in real time. We found that the SARS-1 and SARS-2 PPs also induce membrane ordering to the extent that separate FPs do, and mutations of the negatively charged residues also significantly suppress the membrane ordering activity. However, the slower kinetics of the FP ordering activity vs. that of the PP suggests the need for initial trimerization of the FPs.
Keywords: Membrane fusion; coronavirus; electron spin resonance; spike glycoprotein; viral entry.