modeling

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.


INTRODUCTION

In order to display xylanase on the yeast surface, the choice of anchor protein is crucial for the beneficial presentation of the target protein, which can determine the application and characteristics of the target protein. Yeast anchor proteins have signal sequences that can guide the transportation of target proteins to the cell surface, thereby anchoring them to the cell wall surface.

Yeast anchor proteins1 can be classified into 3 categories according to the different mechanisms of embedding proteins and attachment to the cell wall, GPI (glycosylphosphatidylinositol), FS/FL (flocculation), and Pir (proteins with internal repeats). For GPI, it provides covalent between the target protein and the cell walls that are glucan β-1 and 6; on the other hand, FS/FL attaches to the cell wall polysaccharides such as mannans via non-covalent bonds, while Pir promotes the covalent linkage between the target protein and the cell walls that are glucan β-1,3 and structural proteins through disulfide bonds.

Of these 3 mechanisms, we chose two of the most common anchor proteins in the yeast surface display system, GPI-modified cell wall protein 61(GCW61)2 and PIR-13 , for anchoring xylanase to the surface of yeast. The problem that might occur after the process of combining the yeast and the xylanase is that the xylanase might lose its enzymatic activity or have reduced activity due to improper folding or interference with its active site and binding site. To solve this problem, we have chosen to use protein structure simulation to predict and obtain the structure and active site of the edited protein.


MODELING TOOLS FOR PREDICTING 3D PROTEIN STRUCTURE AND STABILITY

I-TASSER

TASSER I-TASSER4,5,6 (Iterative Threading ASSEmbly Refinement) is a computational method used for predicting the 3D structure and functions of proteins from their amino acid sequences. It identifies potential structural templates from the Protein Data Bank database and constructs full-length 3D models by reassembling template fragments through iterative simulations. The final model is determined by clustering the generated models based on structural similarities, with the centroid of the most populated cluster chosen as the predicted 3D structure. Each model is assigned a C-score, indicating the confidence level of the prediction.

YASARA with FoldX plugin

YASARA7/FoldX8 is employed to calculate the stability of each protein variant, expressed in kcal/mol. This tool helps in understanding the structural integrity and potential functional efficiency of the protein under different conditions. FoldX, integrated within the YASARA suite, allows for precise energy calculations and stability assessments. By analyzing the free energy changes (ΔΔG) of mutations, YASARA/FoldX provides insights into how structural modifications might affect the overall stability and functionality of the protein, aiding in the design of more stable and efficient variants.

PyMOL

PyMOL9 is a molecular visualization tool used to generate and visualize 3D structure images of protein variants. It allows for detailed observation of molecular architecture, facilitating the identification of potential functional sites and structural features. By importing the PDB-formatted models generated by I-TASSER, PyMOL enables researchers to color-code secondary structures, highlight significant residues, and examine the spatial relationships within the protein. This detailed visualization aids in the interpretation of structural data and the communication of complex molecular information.


Xylanase Constructs

Xylanase-WT

Xylanase-GCW61

Xylanase-Pir1


RESULT

Table 1 | Comparison of Xylanase Variants in Terms of Ligand Binding Sites, Active Sites, Stability, and 3D Structures

* Ligand binding site residues and active site residues predicted by I-TASSER with C-SCORE representing a confidence score for estimating the quality of predicted models.

** Stability in terms of free energy (kcal/mol) predicted by YASARA with FoldX plugin using models from I-TASSER

*** Protein 3D structure output generated by PyMOL using models from I-TASSER

Figure 1 | (A) Xylanase (B) Xylanase-GCW61 (C) Xylanase-Pir1.The 3D protein models were generated by I-TASSER and imported into PyMOL for visualization. The models are colored by secondary structures: turquoise for alpha-helices, purple for beta-sheets, and pink for unstructured or flexible loops. Sphere colors: blue for GS linkers, and red for either GCW61 or Pir1 anchor proteins. Glowing residues highlight: yellow for the predicted active sites, and green for the original catalytic triad.

Xylanase from Streptomyces thermovulgaris has a conserved catalytic dyad consisting of two glutamate (Glu) residues (E82-E171), similar to other xylanases11,12. We modeled wild-type xylanase (Xylanase-WT), Xylanase-GCW61, and Xylanase-Pir1 using I-TASSER to predict their 3D structures. The active site residues in Xylanase-WT matched published data, with residues 82 and 171 showing high confidence scores (C-SCORE: 0.637). The stability of Xylanase-WT was calculated to be 182.1 kcal/mol.

For Xylanase-GCW61, the ligand binding sites were consistent with the wild-type, but the active site residues showed a lower confidence score (C-SCORE: 0.459). The stability decreased to 351.7 kcal/mol. Similarly, Xylanase-Pir1 exhibited different ligand binding sites and a lower confidence score for the active site residues (C-SCORE: 0.218), with the lowest stability of 951.93 kcal/mol.

These findings indicate that while Xylanase-GCW61 and Xylanase-Pir1 maintain the essential active sites, their overall stability is compromised compared to the wild-type. This suggests potential challenges in protein expression levels and stability, which require further experimental validation.


MEASUREMENT

Using Experimental Data to Validate and Develop the Xylanase-GCW61 Model

To develop and validate our model, we used precise measurements of the xylanase-GCW61 enzyme and its mutants. We created mutants of Xylanase-GCW61 (E82A and E171A) to address the predicted catalytic glutamate residues' role in xylanase activity, with the assistance of Prof. Hung-Jen Liu at National Chung Hsing University. DNS assays were conducted to quantify the enzyme activity, as detailed in our ENGINEERING and MEASUREMENT pages. The results showed in Table 2 that the activity levels of Xylanase-GCW61 (E82A) and Xylanase-GCW61 (E171A) were below the detection threshold, in contrast to the high activity of wild-type Xylanase-GCW61 at 106±14 Unit/mL/min. These measurements were crucial for validating our model, as they confirmed the essential role of the catalytic dyad in xylanase activity. This empirical data directly influenced the accuracy and reliability of our 3D structural models and stability predictions, ensuring that our computational predictions aligned with observed enzymatic functions. These findings are consistent with published research on xylanase engineering13,14, thereby reinforcing the robustness of our model.

Table 2 | Comparison of Xylanase Variants in Terms of Ligand Binding Sites, Active Sites, Stability, and 3D Structures

*Note: N.D. indicates no detectable activity.


CONCLUSION

In our study, we utilized computational tools such as I-TASSER, YASARA with FoldX, and PyMOL to model the 3D structures and assess the stability of xylanase variants from Streptomyces thermovulgaris. Xylanase-GCW61 and Xylanase-Pir1 were modeled and compared against wild-type xylanase. Despite maintaining essential active sites, the stability of the engineered variants was compromised. Experimental validation using DNS assays confirmed the critical role of the catalytic dyad in xylanase activity. Our findings highlight the importance of selecting suitable anchor proteins for functional enzyme display in yeast systems, while also addressing challenges related to protein stability.


REFERENCES

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