Wang Chenghai, Li Min, Nan Xiaoyan, Deng Yang, Fan Shilong, Lan Jun
School of Biomedical Sciences, Hunan University, Changsha, China.
Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.
Protein Sci. 2025 May;34(5):e70117. doi: 10.1002/pro.70117.
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.
2013年在中菊头蝠中发现了RaTG13冠状病毒，其与严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的基因组同源性为96.3%，这表明蝙蝠是SARS-CoV-2的起源。虽然已知人类血管紧张素转换酶2（hACE2）和中菊头蝠ACE2（bACE2-Ra，有7种多态性变体，命名为01-07）均作为这些冠状病毒的进入受体，但RaTG13受体结合域（RBD）与bACE2-Ra的结合机制仍知之甚少。在此，我们发现与SARS-CoV-2 RBD（372 nM）相比，可以较低亲和力（2.42 μM）与bACE2-Ra-07结合。RaTG13的N370残基处额外的糖基化对RaTG13 RBD与bACE2-Ra-07的结合影响不大。解析了与bACE2-Ra-07结合的SARS-CoV-2和RaTG13 N370Q RBD的晶体结构。界面分析和表面等离子体共振（SPR）测定表明，493、498、501和505位的残基取代可能在SARS-CoV-2 RBD对bACE2-Ra-07的跨物种识别中发挥更重要作用。此外，N370Q突变使从广西分离的穿山甲冠状病毒（PCoV-GX）的RBD与bACE2-Ra-07受体之间的结合亲和力提高了10倍以上。此外，最近流行的SARS-CoV-2变体RBD广泛保留了与bACE2-Ra-07受体的相互作用。我们的研究结果为SARS-CoV-2的跨物种进化提供了新的线索，并促使人们迫切需要监测蝙蝠中冠状病毒的传播情况，以更好地预防未来的溢出事件。
