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. 2021 Aug 25;143(33):13205-13211.
doi: 10.1021/jacs.1c05435. Epub 2021 Aug 10.

Fusion Peptide of SARS-CoV-2 Spike Rearranges into a Wedge Inserted in Bilayered Micelles

Rama K Koppisetti  1 , Yan G Fulcher  1 , Steven R Van Doren  1   2
Affiliations

Fusion Peptide of SARS-CoV-2 Spike Rearranges into a Wedge Inserted in Bilayered Micelles

Rama K Koppisetti et al. J Am Chem Soc. .

Abstract

The receptor binding and proteolysis of Spike of SARS-CoV-2 release its S2 subunit to rearrange and catalyze viral-cell fusion. This deploys the fusion peptide for insertion into the cell membranes targeted. We show that this fusion peptide transforms from intrinsic disorder in solution into a wedge-shaped structure inserted in bilayered micelles, according to chemical shifts, 15N NMR relaxation, and NOEs. The globular fold of three helices contrasts the open, extended forms of this region observed in the electron density of compact prefusion states. In the hydrophobic, narrow end of the wedge, helices 1 and 2 contact the fatty acyl chains of phospholipids, according to NOEs and proximity to a nitroxide spin label deep in the membrane mimic. The polar end of the wedge may engage and displace lipid head groups and bind Ca2+ ions for membrane fusion. Polar helix 3 protrudes from the bilayer where it might be accessible to antibodies.

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Figures

Figure 1.
Figure 1.. Subunits, domains, and fusion peptide sequence of SARS-CoV-2 Spike.
The sequence of the fusion peptide (FP) of this study is shown in red. Its nomenclature in other studies is labeled with gray text.
Figure 2.
Figure 2.. Transformation of prefusion S to mature S2 at a hemifusion stalk.
Maturation of S2 frees FP for membrane insertion and fusion. The structures of postfusion S2, ACE2, an ACE2-S complex, and the TM domain are from refs -, respectively.
Figure 3.
Figure 3.. The fusion peptide of SARS-CoV-2 is disordered in aqueous solution but helical in bicelles.
The free state is represented by red squares and the state bound to bicelles by blue circles. The bicelles were composed of 120 mM DH7PC and 60 mM DMPC. NMR spectra were measured at pH 5.0, 32 °C, 800 MHz. (A) The 15N TROSY free in solution is superposed with that in bicelles. (B) 15N NMR relaxation rate constants R2 and R1 are plotted as a ratio. (C) The secondary 13Cα and 13Cβ chemical shifts are plotted as a difference. (D) The bicelle-induced disorder-to-order transition is accompanied by large shifts of the backbone amide NMR peaks, calculated as radial changes using eq. S1 in SI.
Figure 4.
Figure 4.. The fusion peptide of SARS-CoV-2 closes into a wedge-shaped structure in bicelles.
The lowest energy model from the ensemble of NMR structures (PDB: 7MY8, model 1) is plotted in a shared orientation with coloring of the chain from blue at the N-terminus to red at the C-terminus. A) Side chains and backbone ribbon are plotted. (B)The solvent-accessible surface is colored by electrostatic potential using APBS Tools 2.1 -.
Figure 5.
Figure 5.. PRE and NOE evidence of FP insertion into bicelles.
About one nitroxide spin-labeled 14-doxyl PC probe per leaflet was added to the bicelles. Proximity to the nitroxide label deep in the bicelles is symbolized with red for strong paramagnetic broadening of the amide NMR line. Gray symbolizes minimal broadening. (A) The PREs are estimated from amide NMR peak heights before and after addition of 14-doxyl PC. The 15N HSQC used for detection was modified to include a 1H PROJECT CPMG train of 8 ms ,. (B) Residues with PREs to amide groups ≥ 0.7 in A are red while those between 0.7 and 0.3 are pink. Side chains that appear to have NOEs to phospholipid acyl chains (Fig. S10) are plotted. (C) A hypothesis regarding insertion of FP in a leaflet of DMPC is shown with lipids symbolized by dots. A faint mesh marks the surface of FP. The CHARMM-GUI Membrane Builder was used.
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