ACERT Highlights: Recent Highlights

National Institute of General Medical Sciences
	National Biomedical Resource for Advanced ESR Spectroscopy

Recent Highlights

Negatively Charged Residues in the Membrane Ordering Activity of SARS-1 and SARS-2 Fusion Peptides

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 Ca²⁺-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 Ca²⁺. 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 Ca²⁺ ions: its two segments FP1 and FP2 each bind one Ca²⁺. The binding of Ca²⁺, the folding of FP (both studied by ITC experiments), and the ordering activity correlate very well across the mutants, suggesting that the Ca²⁺ 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.

Publication: Biophys. J. 121, 207-227 (2022); PMC8683214.

Figure: Time-dependent ESR experiments in the pseudotype viral particle (PP) - SUV docking system. A) the schematic diagram of the PP-SUV docking system. The fusion protein (in this case EBOV G protein or SARS-1 S protein) were expressed and assembled as trimers on the PP membrane. DPPTC was incorporated in POPC/POPS/Chol=3/1/1 SUV. The PP and SUV were mixed at a PP:SUV ratio of ca. 1:5, and triggered by the addition of 2 mM Ca²⁺. (B) the plot of local order parameters of DPPTC changes (ΔS₀) during the time course of measurement. Black, EBOV PP; red, SARS-1 PP; blue, MERS PP; green, empty PP (no viral glycoprotein expressed). (C) Same as (B) using SARS-1 PP and SARS-2 PP in different triggering conditions. Black, SARS-1 PP triggered by 2mM Ca²⁺; red, by 2mM Mg²⁺; blue, control by injecting the same volume of buffer; green, SARS-1 PP triggered by 2mM Ca²⁺; purple, by 2mM Mg²⁺. (D) Same as (B) using wildtype (WT) SARS-1 PP (black), D830A mutant (red), and D825A_D830A double mutant (blue). (E) comparing the ΔS₀ induced by separated FP and PP. (F) comparing the time course of SARS-1 PP (black) and SARS-1 FP (red). In the FP case, we mixed the separate FP and the SUV: the amount of the SUV is the same as the one used in the PP case, and the amount of the FP is 1% of the amount of lipids. The PP has an early start and a steeper slope, while the FP has a late start and a flatter slope although they reach almost the same final ΔS₀.

Alex L. Lai, Jack H. Freed. (ACERT)

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ACERT is supported by grant 1R24GM146107 from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

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