Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein

Abstract

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathology. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains tightly bind the essential free fatty acid linoleic acid (LA) in three composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.

Fig. 1. Cryo-EM structure of the SARS-CoV-2 S linoleic acid complex.

(A) Cryo-EM density of the S trimer is shown (left). Monomers are in cyan, green, and pink, respectively. The structure is also shown in a cartoon representation in a front view (middle) and top view (right). Bound LA is illustrated as orange spheres. One LA binding pocket is surrounded by the red box. (B) Composite LA binding pocket formed by adjacent RBDs. Tube-shaped EM density is shown. (C) LC-MS analysis of purified S. (Top) Chemical structure and molecular weight (MW) of LA. (Middle) C4 column elution profile. (Bottom) Electrospray ionization time-of-flight (ESI-TOF) spectra of wash solution (gray) and C4 peak elution fraction (black), with peak molecular weight indicated. Intens., intensity, m/z, mass/charge ratio. (D) Hydrophobic LA binding pocket in a surface representation, illustrating the excellent fit of bound LA (orange; shown in ball-and-stick representation). Blue and red indicate positive and negative surface charge, respectively. (E) LA interactions with amino acids in the binding pocket. The acidic LA headgroup is in the vicinity of an arginine (Arg⁴⁰⁸) and a glutamine (Gln⁴⁰⁹).

Fig. 2. Functional characterization of LA-bound SARS-CoV-2 S.

(A) Insect cell (Hi5)–expressed S (dark blue bars), insect cell–expressed RBD (light blue bars), and mammalian (HEK293)–expressed S (white bars) in competition ELISAs with immobilized ACE2. Error bars indicate SDs (three replicates). A, absorbance; Conc., concentration. (B) Interaction of LA-bound SARS-CoV-2 S protein with ACE2 was analyzed by SEC, evidencing complex formation. (Left) SEC profiles are shown for ACE2 (yellow, III), LA-bound S (green, II), and a mixture of ACE2 and LA-bound S (orange, I). (Right) Peak fractions (I to III) were analyzed by SDS–polyacrylamide gel electrophoresis, evidencing the expected proteins. (C) (Left) Top view of the LA-bound S glycoprotein trimer, with RBDs shown in cyan, green, and pink. In each RBD subunit, the motif responsible for ACE2 binding (RBM) is in red, and LA is shown as orange spheres. (Right) A close-up view into the cyan RBD shows that the RBM is fully ordered and that LA and RBM are not in direct contact. (D) SPR analysis of binding of the LA-bound S trimer (orange curves) and the apo S trimer (green curves) to immobilized ACE2. Apo S and LA-bound S were diluted to concentrations of 40 and 160 nM, respectively. Black lines correspond to a global fit from a 1:1 binding model. RU, resonance units. (E) Synergistic effect of LA and remdesivir on SARS-CoV-2 replication. Effects of varying doses of remdesivir ± 50 μM LA on virus infection are shown. Human Caco-2 ACE2+ cells were infected with SARS-CoV-2 and then treated with varying doses of remdesivir ± 50 μM LA. At 96 hours after infection, cells were fixed and infected cells were detected by immunofluorescence assay using an anti-N antibody (green). Cell nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI; blue). Representative images corresponding to the remdesivir dose range of 20 to 200 nM are shown. (F) Amount of extracellular virus present in wells (n = 3) at the dose combinations shown was determined by quantitative reverse transcription polymerase chain reaction (performed in duplicate for each sample). Error bars show SD.

Fig. 3. Comparison of LA-bound and apo S structures.

(A) Superimposition of LA-bound SARS-CoV-2 RBD (cyan) and ligand-free apo RBD (gray) [PDB ID 6VXX (7)]. The gating helix at the entrance of the hydrophobic pocket moves by 6 Å in the presence of LA. Tyr³⁶⁵ and Tyr³⁶⁹ swing away, avoiding clashes with LA (orange). Black arrows indicate the rearrangements. (B) Same structure as in (A) rotated by 90° as indicated, showing the entrance of the hydrophobic pocket. (C) Formation of a composite LA binding pocket by two adjacent RBDs in LA-bound S involves a ~5-Å movement of RBD2 (green) toward RBD1 (cyan) as opposed to apo S (gray). (D) Superimposition of the RBD trimer of apo S (gray) and LA-bound S (RBD1, cyan; RBD2, green; RBD3, pink; LA, orange) is shown (left). The individual RBD trimers are depicted for LA-bound S (right, top) and apo S (right, bottom), with RBDs boxed in black, highlighting the compaction of RBDs in the LA-bound S structure.

Fig. 4. Human coronavirus RBD architectures.

(A) Alignments of the seven CoV strains that can infect humans, highlighting conserved residues. Cyan, residues lining the hydrophobic pocket; purple, gating helix residues; green, residues positioned to interact with the LA polar headgroup; and red, Glu³⁷⁵ in HKU1 [see panel (E)]. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. (B) Superimposition of RBD1 of LA-bound SARS-CoV-2 (RBD2, green) with RBD1 of ligand-free apo SARS-CoV [RBD1 and RBD2, magenta; PDB ID 5X58 (19)] indicates a conservation of the composite binding pocket. (C) Superimposition of RBD1 of LA-bound SARS-CoV-2 (RBD2 is omitted for clarity) with RBD1 of MERS-CoV [RBD1 and RBD2, forest green; PDB ID 5X5F (19)]. (D) Superimposition of RBD1 of LA-bound SARS-CoV-2 (RBD2, green) with RBD1 of OC43 [RBD1 and RBD2, purple; PDB ID 6NZK (18)]. (E) Superimposition of LA-bound SARS-CoV-2 RBD with HKU1 RBD [brown; PDBID 5GNB (26)]. LA is omitted in SARS-CoV-2 RBD for clarity.