SARS-CoV-2 infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and multivalent interactions play critical roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2-5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate in comparison to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a novel way to investigate the roles of mutations and blocking agents for targeted pharmaceutical intervention.

A Tethered Ligand Assay to Probe SARS-CoV-2:ACE2 Interactions