Molecular dynamics and free energy studies on the carboxypeptidases complexed with peptide/small molecular inhibitor: mechanism for drug resistance.

As one potent plant protease inhibitor, potato carboxypeptidase inhibitor (PCI) can competitively inhibit insect digestive metallocarboxypeptidases (MCPs) through interfering with its digestive system that causes amino acid deficiencies and leading to serious developmental delay and mortality. However, this effective biological pest control is significantly impaired by the PCI-resistant insect MCPs. Therefore, deep understanding of the resistant mechanism of insect MCPs is particularly necessary for designing new durable pest control regimen and developing effective pesticides. In this study, the binding of PCI and small molecular inhibitor THI to insect PCI-sensitive/-resistant MCPs and human MCP was investigated by docking, molecular dynamics (MD) simulations and thermodynamic analysis. The structural analysis from MD simulations indicates that the PCI-resistant mechanism of CPBHz is mainly dominated by the Trp277A, which changes the conformation of β8-α9 loop and therefore narrow the access to the active site of CPBHz, prohibiting the entrance of the C termini tail of PCI. Additionally, the insertion of Gly247A weakens the stabilization of CPBHz and PCI through disrupting the hydrogen bond formation with its surrounding residues. Furthermore, the predicted binding free energies gives explanation of structure affinity relationship of PCI and THI with MCPs and suggest that the electrostatic energy is the main contribution term affecting the difference in binding affinities. Finally, the decomposition analysis of binding free energies infers that the key residues Glu72, Arg127, Ile247/Leu247 and Glu270 are critical for the binding of PCI/THI to MCPs.