Epistatic shifts rationalize mutational constraints of SARS-CoV-2 receptor-binding domain

In a recent study posted to the bioRxiv* pre-print server, researchers used deep mutational scans to measure the effect of each amino-acid mutation in the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variants.

Study: Shifting mutational constraints in the SARS-CoV-2 receptor-binding domain during viral evolution. Image Credit: Mediantone/Shutterstock
Study: Shifting mutational constraints in the SARS-CoV-2 receptor-binding domain during viral evolution. Image Credit: Mediantone/Shutterstock

Mutations in the SARS-CoV-2 spike (S) protein, specifically within RBD, not only impact its affinity for host angiotensin-converting enzyme 2 (ACE2) receptor and recognition by antibodies; but additionally, via a phenomenon called epistasis, which could modulate the effects of mutations at other sites for viral evolution.

Previously, the authors used the native dimeric ACE2 to experimentally measure the impact of all single amino-acid mutations on the ACE2-binding affinity of the ancestral Wuhan-Hu-1 RBD and inform SARS-CoV-2 evolution surveillance. They identified the N501Y mutation had an enhancing effect on ACE2-RBD binding before this consequential mutation emerged in the SARS-CoV-2 Alpha variant.

About the study

Building on the findings of their previous work, in this present study, researchers attempted to systematically understand how epistasis shifts the effects of the SARS-CoV-2 RBD mutations.

They constructed site-saturation mutagenesis libraries of the RBD of ancestral Wuhan-Hu-1 and four other SARS-CoV-2 variants, including Alpha (N501Y), Beta (K417N+E484K+N501Y), Delta (L452R+T478K), and Eta (E484K), cloned these into a yeast-surface display platform and examined the impact of every amino-acid mutation on ACE2-binding affinity and yeast surface-expression levels via fluorescence-activated cell sorting (FACS) and high-throughput sequencing.

In place of native ACE2 dimer, they used monomeric ACE2 ectodomain to measure 1:1 ACE2-binding affinities. It helped provide more granular information about affinity-enhancing effects.

Further, using molecular dynamic (MD) simulations, the researchers examined crystal structures of the ACE2-bound RBD to structurally establish the underlying causes of epistatic shifts and their impact on SARS-CoV-2 RBD mutations.


Consistent with their previous findings, the N501Y mutation caused epistatic shifts that impacted RBD mutations that altered ACE2-binding affinity. The effect of the N501Y mutation in the Delta and Eta RBDs was similar to those in the ancestral Wuhan-Hu-1 RBD; however, the Alpha RBD, containing N501Y mutation only, reiterated the differences in the Beta RBD.

In the Beta RBD, a unique epistatic shift in the effect of mutations to serine or threonine at site 419 also introduced an N-linked glycosylation sequon when the K417N mutation presented an asparagine.

The authors categorized epistatic shifts due to N501Y in RBD sites into three structural groups. 

First was the epistatic effect at the direct N501-contact residue Q498, plus epistatic shifts at sites 491-496. The second category covered sites 446, 447, and 449, which exhibited epistatic shifts in the presence of N501Y but were spatially adjacent to residue 498. The last and third category covered sites that epistatically shifted due to N501Y and covered residue R403, plus several residues forming an interaction network linking site 501 to 403.

Although N501Y improves RBD-ACE2 binding by 15-fold, in conjunction with Q498R, it confers an additional 25-fold enhancement in ACE2 affinity. The affinity buffer provided by the epistatic Q498R/N501Y pair enables the RBD of the Omicron BA.1 and BA.2 variants to tolerate other mutations that are deleterious for ACE2 binding but contribute to antibody escape. Introducing R498Q and Y501N reversions into the Omicron BA.1 S-pseudotyped lentiviral particles further validated these affinity measurements and demonstrated that all Omicron RBD mutations (except Q498R and N501Y) are deleterious.

The crystal structure of the ACE2-bound Beta RBD, observed at 2.45Å resolution and compared to previously determined ACE2-bound Wuhan-Hu-1 and Beta variant structures, did not reveal structural perturbations that explained epistatic shifts between the Wuhan-Hu-1 and Beta RBDs. However, the K417N, E484K, and N501Y substitutions were found to cause minimal changes to the Beta RBD backbone. Crystal structure analysis revealed that epistatic shifts occurred although the global and local RBD structures remain conserved. 

MD simulations of crystal structures of ACE2 bound Wuhan Hu-1, Omicron RBDs showed that while the Wuhan-Hu-1 featured a stable polar contact network between ACE2 residues D38 and K353 and RBD residue Q498, the N501Y substitution in Omicron RBD disrupted it.

In the Omicron structure, the larger Y501 sterically blocked K353 from forming the contact bridging D38 and RBD residue 498. In lieu, D38 alternated salt bridge contacts with K353 and substituted the R498 side chain. Therefore, N501Y potentiated the affinity enhancement of Q498R by improving the energetic benefit of the R498:D38 salt bridge when D38 could no longer jointly coordinate K353 in this conformation.


To conclude, SARS-CoV-2 is constantly mutating to shape potential routes to evolve by shifting the effects of key substitutions. In this context, the present study provided a large-scale sequence-function map that could aid in understanding how SARS-CoV-2 and its variants might remodel the amino-acid sequences of their RBDs while retaining high ACE2 affinity, as done by other human coronaviruses.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Neha Mathur

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.


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