Fig 1.
Phylogenetic tree of TGEV and PRCV strains (isolates) built based on the S-protein amino acid sequences. The phylogenetic tree was built using the Maximum Likelihood method and the JTT matrix-based model based on the results of multiple alignment of S-protein amino acid sequences performed according to the MUSCLE algorithm.
Fig 2.
Comparison of amino acid sequences of the S-protein RBD regions among TGEV, PRCV, and other coronaviruses.
Conservation of amino acid substitutions in TGEV and PRCV strains was assessed using Grantham’s distances: conservative (green, 0–50), moderately conservative (yellow, 51–100), moderately radical (orange, 101–150), and radical (red, ≥ 151). Identical substitutions in other coronaviruses were similarly color-coded. The RBD region spans residues 522–671 in the reference TGEV Purdue strain. Residue S522 is omitted due to a gap in multiple alignment with sequences from other coronavirus species; details on substitutions and Grantham’s distances are provided in S2 Table.
Table 1.
Distance matrix between S-protein amino acid sequences of 16 TGEV and PRCV strains.
Table 2.
ΔG (kcal/mol) calculations for complexes of receptor-binding domains of TGEV strains with APN receptors.
Table 3.
ΔG (kcal/mol) calculations for complexes of receptor-binding domains of PRCV strains with APN receptors, kcal/mol.
Table 4.
Prediction on the ΔΔG and ΔG (kcal/mol) of the complexes, involving pAPN and receptor-binding domain of the TGEV 133 with single mutations.
Table 5.
Prediction on the ΔΔG and ΔG (kcal/mol) of the complexes, involving hAPN and receptor-binding domain of the TGEV 133 with single mutations.
Table 6.
ΔG (kcal/mol) calculations for complexes involving TGEV 133 receptor-binding domain with double mutations.
Fig 3.
Three-dimensional structure of the RBD of TGEV 133 strains in complexes with pAPN and hAPN: general view of complex with pAPN (A), interfaces of the complexes with pAPN (B) and hAPN (C).
The structure of APN is depicted in green, while the RBD is shown in magenta. The positions of mutations in the TGEV 133 strain relative to the reference Purdue strain are highlighted in cyan, and the positions of theoretical mutations that may stabilize the complex are indicated in blue. Length of H-bonds is indicated in angstrom (Å).
Fig 4.
Analysis of molecular dynamics trajectories.
(A–B) Root mean square deviation (RMSD) of complexes involving pAPN (A) and hAPN (B). (C–D) Root mean square fluctuation (RMSF) of pAPN (C) and hAPN (D). (E–F) RMSF of RBDs of virus strains in complexes involving pAPN (E) and hAPN (F). (G–H) Radius of gyration (Rg) of complexes involving pAPN (G) and hAPN (H). (I–J) Solvent-accessible surface area (SASA) of complexes involving pAPN (I) and hAPN (J). Graphs for complexes involving the TGEV Purdue RBD are shown in blue, the TGEV 133 RBD in green, and the TGEV 133 RBD with additional mutations in yellow. Residue numbering in panels (E) and (F) follows that of the Purdue strain. The raw data used in the analysis of RMSD, RMSF, Rg, and SASA are provided in S6 Table.
Fig 5.
Analysis of virus binding to APN through molecular dynamics.
(A–B) H-bonds between RBD and APN in complexes involving pAPN (A) and hAPN (B). (C–D) Non-polar contacts between RBD and APN in complexes involving pAPN (C) and hAPN (D). (E–F) Molecular mechanics energy in the gas phase (ΔGgas) for complexes involving pAPN (E) and hAPN (F). (G–H) Solvation energy (ΔGsolv) for complexes involving pAPN (G) and hAPN (H). (I–J) Binding free energy (ΔGtotal) for complexes involving pAPN (I) and hAPN (J). Graphs for complexes involving the TGEV Purdue RBD are shown in blue, the TGEV 133 RBD in green, and the TGEV 133 RBD with additional mutations in yellow. The raw data used in the analysis of H-bonds, non-polar contacts, and the components of binding free energy are provided in S6 Table and S7 Table.
Table 7.
Binding free energy (ΔG, kcal/mol) and its components calculated from conformational states of RBD-APN complexes along molecular dynamics trajectories.
Fig 6.
Dynamics of energy components (ΔE, kcal/mol) that contribute to the binding free energy.
(A–B) Van der Waals interaction energy (ΔEvdW) for complexes involving pAPN (A) and hAPN (B).(C–D) Electrostatic interaction energy (ΔEeel) for complexes involving pAPN (C) and hAPN (D). (E–F) Polar solvation energy calculated using Poisson-Boltzmann method (ΔEpb) for complexes involving pAPN (E) and hAPN (F). (G–H) Non-polar solvation energy (ΔEnpolar) for complexes involving pAPN (G) and hAPN (H). Graphs for complexes involving the TGEV Purdue RBD are shown in blue, the TGEV 133 RBD in green, and the TGEV 133 RBD with additional mutations in yellow. The raw data used in the analysis of the components of binding free energy are provided in S7 Table.
Fig 7.
Principal component analysis (PCA) and Free energy landscape (FEL) analysis.
(A) PCA plot for complexes involving pAPN and RBDs of all three TGEV strains (TGEV Purdue, TGEV 133, and TGEV 133 with additional mutations). (B–D) PCA plots for complexes involving pAPN and RBDs of TGEV Purdue (B), TGEV 133 (C), and TGEV 133 with additional mutations (D). (E–G) FEL plots for complexes involving pAPN and RBDs of TGEV Purdue (E), TGEV 133 (F), and TGEV 133 with additional mutations (G). (H) PCA plot for complexes involving hAPN and RBDs of all three strains. (I–K) PCA plots for complexes involving hAPN and RBDs of TGEV Purdue (I), TGEV 133 (J), and TGEV 133 with additional mutations (K). (L–N) FEL plots for complexes involving hAPN and RBDs of TGEV Purdue (L), TGEV 133 (M), and TGEV 133 with additional mutations (N). Dots corresponding to complexes involving the Purdue RBD are shown in blue on the PCA plots, those involving the TGEV 133 RBD in green, and those involving the TGEV 133 RBD with additional mutations in yellow. On the FEL plots, conformations with the lowest Gibbs energy are marked in blue, while those with the highest Gibbs energy are marked in red.