PT  - JOURNAL ARTICLE
AU  - Fang Tian
AU  - Bei Tong
AU  - Liang Sun
AU  - Shengchao Shi
AU  - Bin Zheng
AU  - Zibin Wang
AU  - Xianchi Dong
AU  - Peng Zheng
TI  - Mutation N501Y in RBD of Spike Protein Strengthens the Interaction between COVID-19 and its Receptor ACE2
AID  - 10.1101/2021.02.14.431117
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.02.14.431117
4099  - http://biorxiv.org/content/early/2021/02/18/2021.02.14.431117.short
4100  - http://biorxiv.org/content/early/2021/02/18/2021.02.14.431117.full
AB  - SARS-CoV-2 is spreading around the world for the past year. Enormous efforts have been taken to understand its mechanism of transmission. It is well established now that the receptor-binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) as its first step of entry. Being a single-stranded RNA virus, SARS-CoV-2 is evolving rapidly. Recently, several variants such as B.1.1.7, B.1.351, and P.1, with a key mutation N501Y on the RBD, appear to be more infectious to humans. To understand its mechanism, we combined cell surface binding assay, kinetics study, single-molecule technique, and computational method to investigate the interaction between these RBD (mutations) and ACE2. Remarkably, RBD with the N501Y mutation exhibited a considerably stronger interaction characterized from all these methodologies, while the other two mutations from B.1.351 contributed to a less effect. Fluorescence-activated cell scan (FACS) assays found that RBD N501Y mutations are of higher binding affinity to ACE2 than the wild type. Surface plasmon resonance further indicated that N501Y mutation had a faster association rate and slower dissociation rate. Consistent with the kinetics study, atomic force microscopy-based single-molecule force microscopy quantify their strength on living cells, showing a higher binding probability and unbinding force for the mutation. Finally, Steered Molecular Dynamics (SMD) simulations on the dissociation of RBD-ACE2 complexes revealed that the N501Y introduced additional π-π and π-cation interaction for the higher force/interaction. Taken together, we suggested that the reinforced interaction from N501Y mutation in RBD should play an essential role in the higher transmission of COVID-19 variants.Competing Interest StatementThe authors have declared no competing interest.(COVID-19)Coronavirus disease-19;(SARS-CoV-2)Severe acute respiratory syndrome coronavirus-2;(RBD)Receptor-binding domain(MERS-CoV)Middle East respiratory syndrome coronavirus;(ACE2)Angiotensin-converting enzyme 2;(SPR)Surface Plasmon Resonance;(FACS)Fluorescence-activated cell Scan;(AFM)Atomic Force Microscopy;(SMFS)Single-molecule force spectroscopy;(SMD)Steered Molecular Dynamics.