Disulfide bonds stabilize the SARS-CoV-2 RBD and preserve binding to ACE2 following thermal denaturation

Abstract The Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates host cell attachment and entry through the binding of its cognate receptor, angiotensin-converting enzyme 2 (ACE2). This interaction occurs within the receptor-binding domain (RBD) of the Spike protein and is critical to the lifecycle and fitness of SARS-CoV-2. A rigorous analysis of this interaction is crucial to understanding this viral entry mechanism. In the present communication, a biophysical examination of the RBD, ACE2, and their interaction is presented. These studies led to the discovery that disulfide bonds stabilize and protect the RBD from thermal and chemical denaturation. It is further hypothesized that disulfide bond stabilization preserves the ability of the RBD to bind ACE2 following its denaturation. The present hypothesis is supported by the finding that the RBD retains ACE2-binding activity after treatment to high temperatures (95°C) under non-reducing, but not reducing, conditions. These results suggest a mechanism by which SARS-CoV-2 overcomes environmental stress to preserve its RBD–ACE2 interaction.