Single-Molecule Analysis of SARS-CoV-2 Binding to C-Type Lectin Receptors.

Despite intense scrutiny throughout the pandemic, development of efficacious drugs against SARS-CoV-2 spread remains hindered. Understanding the underlying mechanisms of viral infection is fundamental for developing novel treatments. While angiotensin converting enzyme 2 (ACE2) is accepted as the key entry receptor of the virus, other infection mechanisms exist. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and its counterpart DC-SIGN-related (DC-SIGNR, also known as L-SIGN) have been recognized as possessing functional roles in COVID-19 disease and binding to SARS-CoV-2 has been demonstrated previously with ensemble and qualitative techniques. Here we examine the thermodynamic and kinetic parameters of the ligand-receptor interaction between these C-type lectins and the SARS-CoV-2 S1 protein using force-distance curve-based AFM and biolayer interferometry. We evidence that the S1 receptor binding domain is likely involved in this bond formation. Further, we employed deglycosidases and examined a nonglycosylated S1 variant to confirm the significance of glycosylation in this interaction. We demonstrate that the high affinity interactions observed occur through a mechanism distinct from that of ACE2.

Atomic force microscopy applied to interrogate nanoscale cellular chemistry and supramolecular bond dynamics for biomedical applications.

Understanding biological interactions at a molecular level grants valuable information relevant to improving medical treatments and outcomes. Among the suite of technologies available, Atomic Force Microscopy (AFM) is unique in its ability to quantitatively probe forces and receptor-ligand interactions in real-time. The ability to assess the formation of supramolecular bonds and intermediates in real-time on surfaces and living cells generates important information relevant to understanding biological phenomena. Combining AFM with fluorescence-based techniques allows for an unprecedented level of insight not only concerning the formation and rupture of bonds, but understanding medically relevant interactions at a molecular level. As the ability of AFM to probe cells and more complex models improves, being able to assess binding kinetics, chemical topographies, and garner spectroscopic information will likely become key to developing further improvements in fields such as cancer, nanomaterials, and virology. The rapid response to the COVID-19 crisis, producing information regarding not just receptor affinities, but also strain-dependent efficacy of neutralizing nanobodies, demonstrates just how viable and integral to the pre-clinical development of information AFM techniques are in this era of medicine.

Molecular insights into receptor binding energetics and neutralization of SARS-CoV-2 variants.

Despite an unprecedented global gain in knowledge since the emergence of SARS-CoV-2, almost all mechanistic knowledge related to the molecular and cellular details of viral replication, pathology and virulence has been generated using early prototypic isolates of SARS-CoV-2. Here, using atomic force microscopy and molecular dynamics, we investigated how these mutations quantitatively affected the kinetic, thermodynamic and structural properties of RBD-ACE2 complex formation. We observed for several variants of concern a significant increase in the RBD-ACE2 complex stability. While the N501Y and E484Q mutations are particularly important for the greater stability, the N501Y mutation is unlikely to significantly affect antibody neutralization. This work provides unprecedented atomistic detail on the binding of SARS-CoV-2 variants and provides insight into the impact of viral mutations on infection-induced immunity.