## 4 Discussion
Molecular studies can reveal critical domains and motifs responsible for VLP function, while ecological interactions might reflect adaptive response mechanisms to pathogens. Comparative genomic studies can elucidate the genetic context of VLP genes, enhancing our understanding of the genetic mechanisms behind these similarities. Thus, the close clustering of the _N. tabacum_ VLP protein sequence with those from other genera suggests potential evolutionary and functional similarities, warranting further investigation into their possible roles and applications. The physicochemical properties of the _N. tabacum_ VLP protein sequence (Table 1) highlight its molecular weight (45.145 kDa), theoretical pI (\(\sim\)10), and stability across various conditions, as indicated by half-life estimates and an aliphatic index of 49.81. The sequence's structural predictions, refined using iTASSER, RoseTTAFold, and AlphaFold, were validated through Ramachandran plots and MolProbity metrics, revealing RoseTTAFold as the most accurate initial model. However, after further refinement using GalaxyRefine, the iTASSER model showed 88.2% of residues in favored regions (RMSD 0.462), while RoseTTAFold excelled with 97.8% in the favored areas (RMSD 0.348). The MolProbity analysis further confirmed RoseTTAFold's superior quality, displaying minimal steric clashes and favorable rotamer conformations. The ProSA tool assessment corroborated these findings, showing that RoseTTAFold (\(-\)4.79 Z-score) closely matches experimentally determined structures, suggesting it is the most accurate VLP model for _N. tabacum_. The functional interaction site prediction of the _N. tabacum_ VLP protein, using CastP 3.0, identified a significant site with an area of 2461.98 A\({}^{2}\) and volume of 2893.06 A\({}^{3}\), involving numerous amino acids (Table 3). Molecular docking studies revealed that VLP binds more strongly to PGN than \(\beta\)-glucan, with binding affinities of \(-\)10.16 kcal/mol and \(-\)7.19 kcal/mol, respectively. Meanwhile, the \(\beta\)-glucan interaction exhibited lower RMSD (1.45 A) than PGN (2.3 A), indicating a more stable binding. Dynamic behavior and normal mode analysis using iMODS highlighted the stability and mobility of the VLP-\(\beta\)-glucan complex, with B-factor values and eigenvalues supporting its stability. These findings suggest a possible interaction between the _N. tabacum_ VLP and \(\beta\)-glucan, which may be relevant to plant defense mechanisms against fungal pathogens and merits further investigation. MDS provided valuable insights into the structural stability, flexibility, and interaction dynamics of VLP with PGN and \(\beta\)-glucan. The observed RMSD for both complexes suggested that VLP undergoes significant conformational changes upon docking with PGN and \(\beta\)-glucan, and it maintains a stable dynamic equilibrium. The VLP-PGN complex exhibited an average RMSD of 13.37 A for the protein, with PGN showing moderate stability, as indicated by an RMSD of 7.3 A. This outcome suggests that PGN adopts a stable binding pose relative to VLP but undergoes slight conformational adjustments during the simulation. Similarly, the VLP-\(\beta\)-glucan complex exhibited an average RMSD of 12.8 A for VLP and 9.52 A for the \(\beta\)-glucan, indicating comparable flexibility and suggesting that both ligands interact with VLP through dynamic binding processes. These RMSD results were consistent with prior research suggesting that RMSD values above 2 A may still reflect meaningful dynamics, especially in protein-ligand interactions [78; 79]. The contact analysis further elucidated the nature of interactions between VLP and the ligands. During the MDS, both PGN and \(\beta\)-glucan formed various interactions with VLP, including hydrogen bonds, water bridges, and hydrophobic interactions, which are essential for ligand binding and stabilization. PGN, in particular, showed a higher frequency of contacts with VLP, with a maximum of 24 total contacts, compared with \(\beta\)-glucan's 18.