Introduction
This modeling work establishes how PETase from Ideonella sakaiensis interacts with PET oligomers while translating those insights into design choices for construct composition and validation; specifically, the IsPETase-Amy3S Coding Part Codon Optimised for Nicotiana benthamiana (BBa_252Q5ZWV).
🎯 Modeling Goals
- Understand PETase–PET Affinity: Determine whether PETase binds PET 1-mer, 2-mer, and 3-mer with sufficient affinity and favourable interaction profiles to justify the project’s enzyme choice and secretion signal strategy.
- Translate Data into Design: Convert docking-derived trends (binding affinity, hydrophobic contacts, hydrogen bonds) into practical design implications and planned bench validation steps.
- Ensure Scientific Rigor: Document assumptions, limitations, and next steps to meet iGEM expectations that a model either guides design or simulates behaviour used in decision-making.
Scope
The modelled construct is based on our simple part featuring PETase from I. sakaiensis and α-amylase 3 signal peptides (BBa_252Q5ZWV), which perform extracellular excretion. By testing its feasibility, we reduce costly lab cycles of cloning, transformation, and plant expression.
Methods Summary
Molecular docking was performed for PET monomer, dimer, and trimer against PETase to extract binding affinity measured in kilocalories per mole (kcal/mol) as well as hydrophobicity and hydrogen-bond maps. In addition, the docking simulation also presents residue-level interaction maps which annotate van der Waals contacts, pi stacking, carbon-hydrogen bonds, conventional hydrogen bonds, and alkyl-pi-donor interactions for each oligomeric substrate bound in the active-site cavity.
The outputs of our model experiment include numeric affinities and qualitative visualisations for interaction patterns and surface hydrophobicity, forming the foundation for our downstream engineering use.
Key Results: Binding the Plastic Challenge
As we explored the molecular dance between Ideonella sakaiensis PETase and PET oligomers, our docking simulations uncovered how this enzyme grips—or gradually releases—its plastic partners.
| Polymer | Binding Affinity (kcal/mol) |
|---|---|
| Mono | -5.3 |
| Di | -5.0 |
| Tri | -4.6 |
Binding affinities (kcal/mol) decreased in magnitude with oligomer size: mono −5.3, di −5.0, tri −4.6, indicating progressively looser binding as polymer length increases. This decrease is likely due to the increase in substrate size.
Visual Insights
The subsequent graphics depict our PETase-PET monomer docking simulation.
Figure 1. Monomer-CPX
Figure 2. Monomer-CPX-Hydrogen Bond.
Figure 3. Monomer-CPX-Hydrophobicity
The subsequent graphics depict our PETase-PET dimer docking simulation.
Figure 1. Dimer-CPX
Figure 2. Dimer-CPX-Hydrogen Bond
Figure 3. Dimer-CPX-Hydrophobicity
Finally, the following graphics depict our PETase-PET trimer docking simulation.
Figure 1. Trimer-CPX
Figure 2. Trimer-CPX-Hydrogen Bond
Figure 3. Trimer-CPX-Hydrophobicity
Qualitative Analysis
PET oligomers consistently docked into the PETase active site with benzene rings stacking and aligning to more hydrophobic regions, suggesting dominant Van der Waals contributions to stabilisation.
In addition, hydrogen bonding increases in quantity alongside oligomer size. However, this increase did not offset the decline in overall affinity, consistent with steric fit limits in the catalytic pocket for longer stacks.
Interpretation: The modelling analyses indicates that prioritising cleavage pathways yielding shorter PET fragments are advantageous, as PETase demonstrates higher catalytic affinity for monomers and dimers compared to trimers within the modelled binding pocket. This finding therefore supports the use of α‑amylase 3 signal peptides, since extracellular secretion increases exposure to PET fragments where short oligomers dominate, consistent with the hydrophobic binding trends. Together with our earlier observation that PET is primarily located in the extracellular domain, these results led to the incorporation of BBa_252Q5ZWV (the modelled construct which contains both PETase and α‑amylase 3 signal peptides) into all constructs used in our plant transformation experiments.