Molecular mechanisms of RaTG13 and SARS-CoV-2 RBD bound to Rhinolophus affinis bat ACE2.

Abstract

The discovery of the RaTG13 coronavirus in Rhinolophus affinis bats in 2013, sharing 96.3% genome homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suggested bats as the origin of SARS-CoV-2. Although both human angiotensin-converting enzyme 2 (hACE2) and Rhinolophus affinis bat ACE2 (bACE2-Ra, seven polymorphic variants named 01-07) are known to serve as entry receptors for these coronaviruses, the binding mechanism of RaTG13 receptor binding domain (RBD) bound to bACE2-Ra remains poorly understood. Here, we found that RaTG13 RBD bound to bACE2-Ra-07 with a weaker affinity (2.42 μM) compared with SARS-CoV-2 RBD (372 nM). Additional glycosylation at residue N370 of RaTG13 had little influence on bACE2-Ra-07 binding by RaTG13 RBD. Crystal structures of the SARS-CoV-2 and RaTG13 N370Q RBD bound to bACE2-Ra-07 were solved. Interface analysis and surface plasmon resonance (SPR) assay indicated that residue substitutions at 493, 498, 501, and 505 may play a more important role in the cross-species recognition of bACE2-Ra-07 by the SARS-CoV-2 RBD. Besides, the N370Q mutation enhanced the binding affinity between the RBD of pangolin coronavirus isolated from Guangxi (PCoV-GX) and the bACE2-Ra-07 receptor by over 10-fold. Furthermore, the recently prevalent SARS-CoV-2 variant RBDs extensively retained the interaction with the bACE2-Ra-07 receptor. Our findings give new lights on the cross-species evolution of SARS-CoV-2 and prompt the urgency to monitor the circulation of coronaviruses in bats to better prevent future spillover.