Substrate-induced changes in dynamics and molecular motions of cuticle-degrading serine protease PL646: a molecular dynamics study

Abstract

Cuticle-degrading serine proteases secreted by nematophagous fungi can degrade the nematode cuticle during the infection processes. PL646, an alkaline cuticle-degrading serine protease derived from the nematophagous fungus Paecilomyces lilacinus, has been shown to have a high nematicidal activity. Although the crystal structure of PL646 provides a solid basis for investigating its structure–function relationship, the detailed aspects of the dynamics involving the substrate binding, orientation, catalysis, product release, and how these processes are regulated, remain unstudied. Molecular dynamics (MD) simulations and metadynamics simulations of PL646 with and without the peptide substrate AAPV were performed to investigate the changes in structure, molecular motions, and free energy landscape (FEL) of PL646 upon substrate binding. The results indicate that during simulations, the substrate-bound PL646 adopts a more stable and compact conformation than the substrate-free form. However, a few regions located opposite the substrate binding pockets or connected to the catalytic residue show increased flexibility upon substrate binding. Combined essential dynamics (ED) analysis reveals that, upon substrate binding, the noticeable displacements occur not only in the substrate binding pockets/sites, but also in the surface-exposed loops. The dynamic pockets caused by the large concerted motions are proposed to be linked to the substrate recognition, binding, orientation, catalysis, and product release of PL646. The constructed FELs reveal that the substrate-free PL646 has a more rugged and wider free energy surface, and a higher minimum free energy level than the proteinase in complex with its substrate, indicating that the substrate binding reduces the conformational flexibility while increasing the stability of PL646. The results presented in this work will facilitate a better understanding of the structure–dynamics–function relationship of the cuticle-degrading serine protease PL646.