Molecular dynamics elucidation of ion-dependent formation mechanisms in lipase hybrid nanoflowers

• Integrated molecular docking and molecular dynamics to investigate lipase hybrid nanoflower formation. • Metal ion binding modulates lipase structural dynamics without compromising global stability. • Ca(II) exhibits higher affinity and a greater number of binding sites on Burkholderia cepacia lipase. • Low metal concentrations enhance lid and facing lid flexibility while preserving catalytic triad stability. • Enzyme surface exposure and conformational mobility correlate with improved hNF catalytic performance. The study aimed to evaluate the influence of metal ion type and concentration on the formation of hybrid nanoflowers (hNFs) from Burkholderia cepacia lipase (BCL), using a computational protocol integrating metal ion-binding site prediction and molecular dynamics simulations. Metal ion-binding sites for Ca(II) and Cu(II) were identified using the MIB server, followed by molecular dynamics simulations to assess conformational stability, flexibility, and surface exposure of the enzyme under low and high metal ion concentrations. The results demonstrated that low metal ion concentrations, which experimentally favored hNFs formation, were associated with controlled increases in flexibility within the central region of the enzyme, particularly in the lid and facing-lid subdomains, while preserving the structural integrity of the catalytic triad. Analyses of RMSF, SASA, and Rg revealed that the Ca(II)-LC system exhibited greater surface exposure and more favorable conformational adjustments compared to the Cu(II)-LC system, aligning with their superior catalytic performance reported in the literature. In contrast, high metal ion concentrations induced more compact enzyme conformations and correlated with the absence of nanoflower formation. Finally, this study provided molecular-level insights into how different metal ions and concentration modulate the structure of BCL during hNFs formation, offering valuable guidance for the design and optimization of hybrid nanoflowers.