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

In two studies, the authors from the UK, Germany and Switzerland, investigated the binding of a free fatty acid to the receptor binding domain (RBD) of the S protein as a distinguishing feature of pathogenic β-coronaviruses. The results showed that a free fatty acidbinding pocket (the Achillesheel”) stabilizes the spike protein in the locked conformation.

The S protein appears to be a major pathogenic factor that contributes to the unique pathogenesis of SARS-CoV-2. It is composed of an S1 subunit and an S2 subunit, separated by host cell proteases. S1 is composed of the N-terminal domain (NTD), the receptor binding domain (RBD) with a receptor binding motif (RBM), and two C-terminal domains. The RBD in the S1 subunit is responsible for attachment to host cells.

In the prefusion conformation, the RBD in the S protein may alternate between closed (down) and open (up) conformations. SARS-CoV and SARS-CoV-2 S require open RBD (up) conformations to interact with the angiotensin-converting enzyme 2 (ACE2) for entry into cells.

About the studies

In their previous work, the same research group discovered that RBD in the S1 subunit includes a free fatty acidbinding pocket, to which a free fatty acid binds. Mass spectroscopy identified this ligand as linoleic acid, an essential omega-6 polyunsaturated fatty acid that the human body is unable to synthesize.

Binding of a free fatty acid stabilizes a locked conformation of the S protein that is incompatible with the binding of ACE2 receptor. Immunofluorescence demonstrated that synthetic mini virus particles decorated with linoleic acid-bound S protein exhibited a decrease in docking to host cells expressing ACE2 as compared to the mini virus particles with a free S protein. These findings confirm that linoleic acid interfered with receptor binding and subsequent host cell entry by stabilizing a locked form of the S protein.

This study on free fatty acid–binding pocket and the spike protein conformation was published in the journal Science. 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)

In another study, the authors investigated whether linoleic acid binding and the functional consequences of linoleic acid binding are conserved in the S proteins of pathogenic β-coronaviruses (β-CoVs): SARS-CoV, MERS-CoV, and SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta, and Omicron) compared to HCoV-HKU1, a β-coronaviruses causing only mild disease (common cold).

Molecular dynamics simulations confirmed the spontaneous binding of linoleic acid to the hydrophobic pockets in the RBD of SARS-CoV, MERS-CoV, and SARS-CoV-2 variants of concern. However, binding was abolished by one mutation in the HCoV-HKU1.

The authors suggested that the conserved free fatty acid-binding pocket in the RBD in the S1 subunit emerges as a hallmark of the S protein structure in pathogenic β-coronaviruses. All SARS-CoV-2 variants of concern maintain this pocket, notably including Omicron, which has accumulated a wide range of mutations in the S protein elsewhere, suggesting that the pocket provides a selective advantage for the virus.

But, the scientists pointed out that the stable binding of linoleic acid to the RBD only occurs after linoleic acid has entered the pocket. It was less clear whether the pocket first has to open by moving the gating helix or whether binding of linoleic acid at the entrance of the pocket can force open the pocket. They found examples for both scenarios in the different simulations, but an opening of the pocket while interacting with linoleic acid (induced fit) occurred more frequently.

In a conclusion, free fatty acid binding to the S protein can be conceived as an Achillesheelof pathogenic β-coronaviruses. This open the possibilities for linoleic acid or linoleic acid mimeticbased antiviral intervention against SARS-CoV-2. Electron tomography of SARS-CoV-2infected cells demonstrated that treatment with linoleic acid inhibited viral replication, resulting in reduced number of deformed virions.

The authors concluded that their investigation of free fatty acid–binding pocket and the spike protein conformation demonstrated that free fatty acid binding is a hallmark of pathogenic β-coronaviruses infection and replication. The interaction between free fatty acid and the S protein, while providing selective advantages to the virus, makes it vulnerable to antiviral intervention by exploiting this highly conserved feature. This could be achieved by supplementing linoleic acid or a related molecule, ideally during the early stages of infection.

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)