To enhance the activity of IsPETase, our team designed two methods. The first method involved adding a hydrophobic domain at the 3' or 5' end of the protein. This hydrophobic domain can bind to the hydrophobic surface of PET, facilitating the interaction between the enzyme and PET, as well as mutations. To predict the structures of the enzyme-domain complex and mutant proteins, we utilized alphafold2. Subsequently, we performed molecular docking using the PET structure file from Pubchem as the substrate. The docking efficiency and activity of the complex were evaluated by calculating the binding free energy using Molecular Mechanics / Poisson Boltzmann (Generalized Born) Surface Area(MM-PBSA).
2.1 Method
Fig. 8 Molecular Docking Flowchart
After preparing the protein and ligand structure files, we imported them into the software. We employed a specified method provided by the software to predict the active sites and performed docking. The structure with the Binding free energy score was considered a reasonable docking structure. Additionally, we selected a water model and used the opls4 force field to perform all-atom energy minimization of the active sites, constructing the simulation system. Binding free energy was then calculated using MM-PBSA.[6].
In terms of interaction determination, we classified the contacts between two atoms as Good, Bad, or Ugly based on a cutoff distance. The cutoff distance, denoted as I, is defined as the ratio of the distance between two atoms to the sum of their van der Waals radii. Contacts with I greater than 1.3 were classified as Good, contacts with I between 1.3 and 0.89 were classified as Bad, contacts with I between 0.89 and 0.75 were classified as Ugly, and contacts with I less than 0.75 were considered non-contact.
2.2 Results of molecular docking
Fig. 9 Complex protein-PET binding free energy calculated by MM-PBSA.(Through literature research, we selected the hydrophobic carbohydrate domain CBM3, as well as a series of carbohydrate domains CBM4, CBM11, LSChi4CBM and LSChi5CBM found in our laboratory preservation cultures.)
The results of the binding free energy calculations revealed that only LSPET4CBM showed an enhancing effect on the enzyme activity, which aligns with our experimental conclusion. Notably, the scores of the ten prepared mutants were generally higher than those of the enzyme-domain complex in terms of binding free energy. This suggests that the activity enhancement obtained through mutation should be higher than that achieved by adding the domain.
Fig. 10 IsPETase mutant-PET binding free energy calculated by MM-PBSA
In our study, WT refers to the wild-type IsPETase, Worse and Better refer to mutants that exhibited consistent results with the experimental data in terms of binding free energy calculation, while Error refers to mutants that did not match the experimental results. Overall, the accuracy of predicting the activity enhancement effect in enzyme engineering through molecular docking and MM-PBSA calculation reached 70%.
Fig. 11 WTIsPETase Activity Pocket
Fig. 12 S93_I94insE Active Pocket
The activity pocket of the mutant A240_S242del underwent a disruptive change. Analyzing the surface of the pocket revealed that compared to the wild-type, the variant exhibited a larger degree of pocket enclosure. From the perspective of the protein-ligand complex, it is evident that the mutation, by reconstructing the active pocket, enhances the binding effect between the protein and the substrate, thereby improving the enzyme activity.
Fig. 13 WTIsPETase-PET
Fig. 14 Q119F-PET
From the perspective of the protein-ligand complex, the mutant's activity pocket fits the ligand better, leading to enhanced binding between the protein and substrate. Specifically, the Q119F mutation reconstructs the active pocket, thereby improving enzyme activity.
Fig. 15 T116R Activated Pocket
Fig. 16 T116R-PET
After the T116R mutation, the residue 116R interacts with 87Y, 88T, and 160S. This interaction results in the formation of a new pocket on the basis of the original activity pocket, with a neutral potential in some parts. Compared to the wild-type, this new pocket better accommodates the methyl end of the PET molecule. In summary, the T116R mutant creates a new pocket-like structure, enhancing the protein's binding to the ligand.
Fig. 17 160S,161M (WTIsPETase) and PET
Fig. 18 160S,161M(W159H) and PET
Tryptophan, being a large amino acid with significant steric hindrance, is mutated to the smaller amino acid histidine. This mutation reduces the spatial distance between 160S, one of the three catalytic groups, and its neighboring residue 161M, bringing them closer to PET. Consequently, there is a significant increase in the number of Good-level contacts represented by green dashed lines after the mutation. Additionally, the hydrogen on the hydroxyl group of 160S in the W159H mutant forms a hydrogen bond with PET, further demonstrating the reduction in steric hindrance between the protein and the ligand caused by the mutation.