Temperature-Dependent Reorganization of Conformational Dynamics and Interaction Networks Underlies Thermostability in...

Polyethylene terephthalate (PET) is one of the most widely used synthetic plastics and a major contributor to global plastic pollution because of its high resistance to degradation. Enzymatic degradation by PET hydrolases (PETase) has emerged as a sustainable strategy for PET recycling; however, the limited thermostability of wild-type PETase restricts its industrial application. To elucidate the molecular basis underlying the different thermal behaviors of PET hydrolases, long-timescale molecular dynamics simulations were performed on WT-PETase, FAST-PETase, and the thermostable cutinase variant LCC-ICCG at 30 °C, 50 °C, and 70 °C. Comparative analyses integrating structural stability, residue flexibility, rigidity networks, free energy landscapes, and neural relational inference models revealed that FAST-PETase and LCC-ICCG exhibited enhanced conformational stability and reduced structural flexibility compared with WT-PETase, particularly under elevated temperatures. The improved thermostability was associated with more compact free energy landscapes, strengthened residue interaction networks, and better preservation of the catalytic architecture during thermal perturbation. These results suggest that an optimal balance between structural rigidity and conformational flexibility is critical for maintaining enzyme stability at elevated temperatures. Overall, this study provides molecular-level insights into the structural determinants of PETase thermostability and offers a theoretical framework for the rational engineering of efficient and heat-resistant plastic-degrading enzymes.