The free fatty acid–binding pocket is a conserved hallmark in pathogenic β-coronavirus spike proteins from SARS-CoV to Omicron

Figures

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  Fig. 1.. Conservation of LA-binding pocket in β-CoVs. (A) Alignments of the B domain of β-CoVs HCoV-HKU1 and HCoV-OC43, and the RBD of SARS-CoV, MERS-CoV, SARS-CoV-2, and VOCs (Alpha, Beta, Gamma, Delta, and Omicron), including the current BA.5 and BA.2.75 variants. Gray box: FFA pocket residues; green: residues in the hydrophilic lid; orange: residue E375 in HCoV-HKU1. (B) Conservation of residues within the domains is shown, calculated with ConSurf (58), mapped on the structure of SARS-CoV-2 RBD (PDB-ID 6ZB5). Orange spheres: LA. (C) Timeline (not proportional) illustrating the emergence of β-CoVs starting with SARS-CoV in 2002 up to SARS-CoV-2 VOC Omicron in 2022. Greek letters, VOC denominations. (D to F) SPR analysis of LA binding to immobilized SARS-CoV RBD (D), MERS-CoV RBD (E), HCoV-HKU1 wild type (WT), and E375A mutant B domain (F), and respective three-dimensional (3D) structures drawn in ribbon presentation. Residues shared with the SARS-CoV-2 RBD are colored in cyan. Purple spheres: E375 in HCoV-HKU1. (G) LA binding to RBDs from the five VOCs analyzed by SPR. LA concentrations ranging from 4 to 10 μM were used for all SPR experiments. For HCoV-HKU1 wild type, only 10 μM was…

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  Fig. 2.. SARS-CoV S adopts LA-bound locked and LA-free open conformations. (A) Cryo-EM density of SARS-CoV S in locked (left) and open (right) conformations, front and top views. Monomers are in cyan, blue, and magenta. RBD-up conformation marked by arrows. (B) Top view of locked S, cartoon representation. Orange spheres: LA. Blue box: fully ordered RBM (blue) and SARS-CoV-2 S RBM (red) (PDB ID 6ZB5). Red boxes: LA-binding pocket formed by adjacent S RBDs. Left: LA’s headgroup interacts with R395 and Q396. Right: Density (mesh) for hydrophobic residues lining the pocket. (C) LA-bound S RBD trimers overlaid with SARS-CoV S, closed conformation without LA (gray) (PDB ID 6ACC). (D) Overlay of S RBD trimers, open and closed conformation (gray) (PDB ID 6ACC). (E) Open conformation, top view. Right: Zoom-in with density for hydrophobic residues lining the pocket (mesh). (F) Overlay of LA-bound and unbound S RBD from locked (blue) and open (gray) conformation. Red arrows: movement of gating helix, Y352 and Y356 upon LA binding. (G) Locked conformation, side view. Red box: region around R620. Close-up: open conformation (gray), residues R620-Y622 and F305 in red. Locked conformation: R620 (blue)…

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  Fig. 3.. MD analysis of LA binding to β-CoV S proteins. (A) LA binding to the hydrophobic pocket of SARS-CoV-2 RBD [PDB ID 6zb5 (11)]. LA (cyan) transiently interacts with the RBD surface before entering the pocket (dark blue). Glycosylation at residues N343 and N331 is shown as spheres. LA positions correspond to first 600 ns of MD simulation shown in (B). Trajectories are colored identically in all panels. (B) Pocket dynamics (gray curve) and LA pocket binding (blue curve) to SARS-CoV-2 RBD (see also fig. S5, A and B). (C) LA binding to SARS-CoV RBD highlighting three different outcomes of simulations (A to C). Right: Position of LA during the simulation (on the surface and in the pocket indicated by # and *, respectively). (D) LA binding to MERS-CoV RBD, three simulations (A to C). (E) Atom-density iso-contour plot derived from accumulated 2.3-μs MD simulation of LA (magenta) bound to MERS-CoV RBD (gray, ribbon and density mesh; cyan, side chains; green, glycosylation groups). LA high atom density is also shown as solid iso-contour volume (magenta). (F) LA binding to HCoV-HKU1 B domain. Left: Four simulation trajectories (A to D). Right: LA binds at the pocket entrance during…

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  Fig. 4.. TEM analysis of GFP-expressing SARS-CoV-2–infected Caco-2-ACE2 cells. Transmission electron microscopy (TEM) images of cells in the absence (A) and presence (B) of treatment with 50 μM LA (added after infection). Left: Overview image of a GFP-SARS-CoV-2–infected cell. Yellow box highlights a region with virions. Scale bars, 500 nm. Right: Close-up view. Scale bars, 200 nm. Green arrows point to virions; LA highlights lipid droplets. (C) Particle quantification and size/shape analysis of virions from green cells. Values are indicated as means ± SD. Significance levels were determined by unpaired t test. ****P < 0.0001. (D) Dose-response inhibition of cPLA2 by LA, compared to cPLA2 inhibitors used as positive controls [Pyr_2 (pyrrolidine-2), AACOCF3, PACOCF3, and EDTA]. Experiments were repeated twice. Data are represented as disintegration per minute [% of maximal enzymatic activity measured in the absence of inhibitor (untreated)].