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The free fatty acid–binding pocket stabilizes SARS-CoV-2 spike protein in its locked conformation (the “Achilles’ heel of SARS-CoV-2”)

Mar 24, 2023 | About the Virus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus. Its genome encodes four structural proteins, namely the spike (S), envelope (E), nucleocapsid (N), and membrane (M) protein. In this study, the consortium of authors from the United Kingdom, Germany, and Switzerland, examined the binding of a free fatty acid to the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein as a distinguishing feature of pathogenic β-coronaviruses (β-CoVs) infection and replication. The results revealed that the free fatty acid-binding pocket stabilizes the SARS-CoV-2 S protein in its locked conformation. 

The S protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. The S protein plays three critical roles in facilitating host cell entry: it must bind the angiotensin-converting enzyme-2 (ACE2) receptor, be proteolytically processed, and promote membrane fusion. The S protein is a glycosylated homotrimer with each monomer composed of subunits S1 and S2. The S1 domain comprises an N-terminal domain (NTD), a receptor-binding domain (RBD) with a receptor binding motif (RBM), and two C-terminal domains. The RBD in the S1 subunit is a short immunogenic fragment responsible for attachment to host cells, mediating the interaction with the human angiotensin-converting enzyme 2 (ACE2) receptor. The S2 subunit contains the fusion machinery required for viral-cell membrane fusion.

Multiple conformations showing RBD in ‘up’ or ‘down’ orientations have been observed by cryo-electron microscopy. In the prefusion conformation, the RBD may alternate between closed (down) and open (up) conformations. The SARS-CoV and SARS-CoV-2 S proteins require an open RBD (up) conformation to interact with ACE2 for host cell entry. 



Free fatty acid-binding pocket stabilizes spike protein

In their previous work, the same research group discovered that RBD contains a free fatty acidbinding pocket, to which a free fatty acid binds. The mass spectroscopy has identified this ligand as linoleic acid, an essential omega-6 polyunsaturated fatty acid, which the human body is unable to synthesize.

A locked conformation of the S protein is incompatible with the binding of the ACE2 receptor, and stabilized by the binding of a free fatty acid. The immunofluorescence findings revealed that synthetic mini virus particles, decorated with linoleic acid-bound S protein, showed decreased docking to host cells expressing ACE2 compared to synthetic mini virus particles with a free S protein. These findings confirmed that linoleic acid interfered with receptor binding and subsequent host cell entry by stabilizing a locked form of the S protein. (Toelzer C, et al. Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein. Science 2020; 370, 725730 (Open Access))

However, the scientists emphasized that stable binding of linoleic acid to the RBD occurs only after linoleic acid has entered the pocket. It is less clear whether the pocket must first be opened by moving the gating helix, or whether the binding of linoleic acid at the entrance of the pocket can force the pocket to open. The scientists found examples for both scenarios in different simulations. However, the opening of the pocket while interacting with linoleic acid (induced fit) occurred more frequently.

According to the authors, the conserved free fatty acid-binding pocket in the RBD appears to be a hallmark of the S protein structure in pathogenic β-CoVs. All SARS-CoV-2 variants of concern maintain this pocket, including Omicron. This suggests that the pocket provides a selective advantage for the virus.

However, the interaction between free fatty acid and the S protein, while providing selective advantages to the virus, renders it vulnerable to antiviral intervention by exploiting this highly conserved feature. Therefore, a free fatty acid binding to the S protein could be considered an “Achilles’ heel” of pathogenic β-CoVs. The results of electron tomography revealed that treatment of cells infected with SARS-CoV-2 with linoleic acid inhibited viral replication and decreased the number of deformed virions.


The authors concluded that the free fatty acidbinding pocket stabilizes the SARS-CoV-2 spike protein in its locked conformation, making it a hallmark of pathogenic β-CoVs infection and replication. These results open up the possibility of using linoleic acid or linoleic acid mimetic–based antiviral interventions against SARS-CoV-2, particularly during the initial stages of infection. 

This article was published in Science Advances.

Journal Reference

Toelzer et al. The free fatty acid–binding pocket is a conserved hallmark in pathogenic β-coronavirus spike proteins from SARS-CoV to Omicron. Sci. Adv. 2022; 8, eadc9179. (Open Access)

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