Impact of SARS-CoV-2 RBM Mutations N501Y and E484K on ACE2 Binding: A Combined Computational and Experimental Study

Abstract

The SARS-CoV-2 spike receptor-binding motif is crucial for viral entry via interaction with the human ACE2 receptor. Mutations N501Y and E484K, found in several variants of concern, impact viral transmissibility and immune escape, but experimental data on their binding effects remain inconsistent. Using isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations, we analyzed the thermodynamic and structural effects of these mutations. ITC confirmed that N501Y increases ACE2 affinity by 2.2-fold, while E484K enhances binding by 5.8-fold. The Beta/Gamma variant (carrying both mutations) showed the strongest affinity, with a 15-fold increase. E484K was enthalpy-driven, while N501Y introduced entropy-driven effects, suggesting hydrophobic interactions and conformational changes. MD simulations revealed distinct binding poses, with Beta/Gamma peptides interacting with a secondary ACE2 site. A strong correlation was found between entropy contributions and hydrophobic contacts. Additionally, a convolutional neural network was used to estimate the free binding energy of these complexes. Our findings confirm that N501Y and E484K enhance ACE2 binding, with the greatest effect when combined, providing insights into SARS-CoV-2 variant evolution and potential therapeutic strategies.

Keywords: ACE2; E484K; ITC; N501Y; SARS-CoV-2; binding affinity; molecular dynamics.

Figure 1

Sequences of the analyzed RBM fragments (peptides) with the single and combined mutations. The variable amino acids are shown in red.

Figure 2

The overall structure of the SARS-CoV-2 RBD bound to the ACE2 receptor. The analyzed RBM fragment is shown in red, with the variable amino acids highlighted in blue. Visualized using UCSF Chimera [21], PDB ID: 6M0J.

Figure 3

Thermodynamic signatures obtained from ITC measurements, detailing the binding interactions between ACE2 and the analyzed RBM fragments of five SARS-CoV-2 variants at pH 7.4 and 25 °C. The Gibbs free energy change (ΔG) is represented in blue, the enthalpy change (ΔH) in green, and the entropy term (−TΔS) in red, with all values expressed in kcal/mol.

Figure 4

Binding of the peptides to glycosylated hACE2. (a) The surface of the hACE2 enzyme at the SARS-CoV-2 spike–ACE2 binding interface. The enzyme is depicted in silver cartoons and the binding surface in a pink wireframe. (a–e) Binding poses of the WT (b), Alpha (c), Beta/Gamma (d), and Zeta (e) RBM peptide fragments. The peptides are depicted in green, red, orange, and blue Van der Waals surface, respectively.

Figure 5

2D LigPlot representation of peptide–ACE2 interactions. Covalent bonds are presented in solid brown lines in the RBM fragments and solid purple lines in the ACE2 receptor. Intermolecular H-bonds are shown in dashed green lines, with participating amino acids noted in green in the peptides and blue in the receptor. Hydrophobic contacts are depicted with red dashed lines and arcs with radiating lines in magenta or red for the ACE2 and the peptides, and the relevant amino acids are denoted in blue and black, respectively.

Figure 6

Correlation between (a) the number of peptide–hACE2 hydrophobic contacts and the experimentally determined entropic contribution to the binding free energy and (b) the number of H-bonds between the peptides and the glycosylated hACE2 and the experimentally measured enthalpy change upon binding. The values for the WT, Alpha, Beta/Gamma, and Zeta RBM fragments are shown in green, red, orange, and blue squares, respectively.

Figure 7

(a) Experimental results for K_D measurement as obtained in publications [14,15,17,23,24,25,26,27,28,29], and this study, numbered in the plot consecutively from 1 to 11 and listed in Supplementary Table S1; (b–d) Summaries of the distribution of the (b) calculated ΔG for the WT and the E484K and N501Y mutations (the dots on the box diagram correspond to our results); (c) D(K_D), and (d) D(ΔG) for the E484K and N501Y mutations.

Figure 8

(a) Theoretical estimations of binding free energy (results from [31,32,33,34], with the last data point representing the CNN prediction from this study); Comparison of the theoretical estimations with experimental data for the (b) binding free energy and (c) relative change in binding free energy.