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

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.