Mechanistic study of the transmission pattern of the SARS-CoV-2 omicron variant

Abstract

The omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) characterized by 30 mutations in its spike protein, has rapidly spread worldwide since November 2021, significantly exacerbating the ongoing COVID-19 pandemic. In order to investigate the relationship between these mutations and the variant's high transmissibility, we conducted a systematic analysis of the mutational effect on spike-angiotensin-converting enzyme-2 (ACE2) interactions and explored the structural/energy correlation of key mutations, utilizing a reliable coarse-grained model. Our study extended beyond the receptor-binding domain (RBD) of spike trimer through comprehensive modeling of the full-length spike trimer rather than just the RBD. Our free-energy calculation revealed that the enhanced binding affinity between the spike protein and the ACE2 receptor is correlated with the increased structural stability of the isolated spike protein, thus explaining the omicron variant's heightened transmissibility. The conclusion was supported by our experimental analyses involving the expression and purification of the full-length spike trimer. Furthermore, the energy decomposition analysis established those electrostatic interactions make major contributions to this effect. We categorized the mutations into four groups and established an analytical framework that can be employed in studying future mutations. Additionally, our calculations rationalized the reduced affinity of the omicron variant towards most available therapeutic neutralizing antibodies, when compared with the wild type. By providing concrete experimental data and offering a solid explanation, this study contributes to a better understanding of the relationship between theories and observations and lays the foundation for future investigations.

Keywords: SARS‐CoV‐2; computational biology; omicron; spike protein.

Figure 1.. The binding free energy change of each mutation of omicron or combinations of all sites of the omicron and delta variant.

The square and line represent the statistics of 15 data points (see Methods); the diamond designates the average value of each square-line (Table S1); the points designate the outliers of the data; the color designates in which region the mutated site is located: N-terminal Domain (blue), Receptor Binding Domain (red), Subdomain 1/2 (green), other unnamed regions (cyan), Heptad Repeat 1 (purple). The text under the X-axis represents the name of mutations or variants, where the red text indicates the mutations with the largest magnitude of $\Delta \Delta \mathrm{G}$.

Figure 2.. The relative magnitude of electrostatic free energy contribution ΔGe=Gomicron−Gwild−type of each charged residue in complex or in isolated spike protein.

The color of points designates the difference of energy contribution $\left(\left|\Delta \Delta {\mathrm{G}}_{\mathrm{e}}\right|=\left|\Delta {\mathrm{G}}_{\text{e}-\text{complex}}-\Delta {\mathrm{G}}_{\text{e}-\text{spike}}\right|\right)$. The shaded region designates all points that do not meet the two criteria of key residues defined in the main-text. The key residues identified are marked by texts. R² = 0.7732.

Figure 3.. Structural representation of the position and the electrical environment of (A) K417N, (B) T547K, (C) N679K, (D) N764K, (E) N856K, and (F) N969K.

The magenta lines and texts indicate the distances of key interactions.

Figure 4.. The experimental results of isolated spike structural stability.

(A)-(F) The thermal stability of spike protein WT and mutants using the nanoDSF method. All spike proteins were in the PBS buffer and the test concentrations were 0.2 mg/mL. The test temperature starts at 20°C and rises 1 degree per minute until it reaches 95°C.the peaks of the first derivative represent the protein started unfolding. (G) The onset temperature of protein unfolding (Temperature_onset) of the isolated wild-type spike protein and mutants. The bar represents the onset temperatures, which were measured by using the nanoDSF method. The data are shown as mean ± SEM based on three experiments. The points and line in blue represent the $\Delta \mathrm{G}2$ of each mutant in our calculations.

Figure 5.. The visualization of spike RBD/ACE2 interface and the experimental binding affinity of RBD/ACE2.

(A) The electrostatic potential surface calculated by APBS of the omicron variant RBD and the wild-type RBD. (B) The locations of N440K, T478K, Q493R, and Q498R in the RBD and the interacting residues of ACE2; Yellow texts marks the charge residues of ACE2, blue texts mark the residues of RBD before mutation, and grey texts marks the residues of RBD after mutation.

Figure 6.. The experimental results of spike/ACE2 binding affinity.

(A)-(F) The biolayer interferometry (BLI) sensorgrams display the binding between ACE2 and spike protein (WT and different mutants). The data are shown as blue lines, and the best fit of the data to a 1:1 binding model is shown in red.(G) K_d values of Spike protein WT and mutants. Equilibrium dissociation constants (K_d) were obtained from k_off/k_on (Table S3). The data are shown as box plots of five repeats (the dots). The center, lower and upper lines in each box indicate the median, the first quartile and the third quartile, respectively. The significances of difference between each mutant and wild-type were performed by single-side Wilcox test. (H) Scatter plot of experimental and calculated $\Delta \Delta {\mathrm{G}}_{\text{binding}}$ for spike protein mutants. Different mutants are represented by different colors. The solid squares “calculate” designates the computational results, while the hollow squares “observe” represent the experimental results. Error bars show the standard error of mean (S.E.M.).