Linoleic Acid-Binding in the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), the agent that causes COVID-19, has produced a prolonged global crisis. Although structures of SARS-CoV-2 are conserved among other members of the Betacoronavirus genus, SARS-CoV-2 displays heightened transmissibility. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein forms a tighter binding interface with the human angiotensin-converting enzyme 2 (ACE2) membrane receptor to initiate infection. Recent cryo-electron microscopy studies of the spike RBD illuminate a potential free fatty acid-binding pocket, which locks the RBD in a closed inactive conformation upon linoleic acid binding. However, the characteristics of this ligand-binding interaction are understudied. We analyzed the linoleic acid-binding pocket through binding and displacement studies that utilize the hydrophobic fluorophore 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). We found that 1,8-ANS binds with a dissociation constant (Kd) of ~70 μM. Further studies will determine the inhibition constant (Ki) of linoleic acid as it competitively displaces 1,8-ANS within the RBD binding pocket to produce a measure of apparent binding affinity. These results will demonstrate the strength of the linoleic acid-binding interaction with the SARS-CoV-2 RBD. We anticipate that these insights may help inform developments of COVID-19 treatments. The free fatty acid-binding pocket may serve as an important target for drugs that aim to prevent the initiation of infection by SARS-CoV-2.