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Updated: Jul 10, 2025
Application of I TASSER, trRosetta, UCSF Chimera, HADDOCK server, and HEX loria for De Novo and In Silico Design of Proteins
Published on: July 8, 2025
In Silico Evaluation, Phylogenetic Analysis, and Structural Modeling of the Class II Hydrophobin Family from
Nahla A Bouqellah1, Peter F Farag2
1Department of Biology, College of Science, Taibah University, P.O. Box 344, Al Madinah Al Munawwarah 42317-8599, Saudi Arabia.
Abstract:
The class II hydrophobin group (HFBII) is an extracellular group of proteins that contain the HFBII domain and eight conserved cysteine residues. These proteins are exclusively secreted by fungi and have multiple functions with a probable role as effectors. In the present study, a total of 45 amino acid sequences of hydrophobin class II proteins from different phytopathogenic fungi were retrieved from the NCBI database. We used the integration of well-designed bioinformatic tools to characterize and predict their physicochemical parameters, novel motifs, 3D structures, multiple sequence alignment (MSA), evolution, and functions as effector proteins through molecular docking. The results revealed new features for these protein members. The ProtParam tool detected the hydrophobicity properties of all proteins except for one hydrophilic protein (KAI3335996.1). Out of 45 proteins, six of them were detected as GPI-anchored proteins by the PredGPI server. Different 3D structure templates with high pTM scores were designed by Multifold v1, AlphaFold2, and trRosetta. Most of the studied proteins were anticipated as apoplastic effectors and matched with the ghyd5 gene of Fusarium graminearum as virulence factors. A protein-protein interaction (PPI) analysis unraveled the molecular function of this group as GTP-binding proteins, while a molecular docking analysis detected a chitin-binding effector role. From the MSA analysis, it was observed that the HFBII sequences shared conserved 2 Pro (P) and 2 Gly (G) amino acids besides the known eight conserved cysteine residues. The evolutionary analysis and phylogenetic tree provided evidence of episodic diversifying selection at the branch level using the aBSREL tool. A detailed in silico analysis of this family and the present findings will provide a better understanding of the HFBII characters and evolutionary relationships, which could be very useful in future studies.
Insights
Class II hydrophobins (HFBII) are fungal effector proteins. This study used bioinformatics to reveal their physicochemical properties, 3D structures, and roles as apoplastic and chitin-binding effectors, uncovering evolutionary insights.
Area of Science:
- Fungal molecular biology
- Bioinformatics
- Protein science
Background:
- Class II hydrophobins (HFBII) are extracellular fungal proteins with diverse functions, potentially acting as effectors.
- These proteins feature the HFBII domain and eight conserved cysteine residues, secreted exclusively by fungi.
- Understanding HFBII proteins is crucial for comprehending fungal pathogenicity and host interactions.
Purpose of the Study:
- To comprehensively characterize 45 hydrophobin class II protein sequences from phytopathogenic fungi using advanced bioinformatics.
- To predict physicochemical parameters, identify novel motifs, elucidate 3D structures, and analyze evolutionary relationships.
- To investigate the functional roles of HFBII proteins as potential effector molecules in fungal pathogenesis.
Main Methods:
- Retrieval of 45 HFBII amino acid sequences from the NCBI database.
- Application of bioinformatic tools (ProtParam, PredGPI, Multifold v1, AlphaFold2, trRosetta, aBSREL) for analysis.
- In silico analyses included physicochemical prediction, 3D structure modeling, Multiple Sequence Alignment (MSA), protein-protein interaction (PPI), molecular docking, and evolutionary analysis.
Main Results:
- Hydrophobicity was a common feature, with one exception. Six proteins were identified as GPI-anchored.
- 3D structures were modeled with high confidence. Most HFBII proteins were predicted as apoplastic effectors, linked to virulence factors like ghyd5.
- MSA revealed conserved Pro and Gly residues alongside cysteines. PPI and docking suggested GTP-binding and chitin-binding roles, respectively. Evolutionary analysis indicated episodic diversifying selection.
Conclusions:
- This study provides a detailed in silico characterization of fungal Class II hydrophobins, revealing novel structural and functional insights.
- Findings highlight the multifaceted roles of HFBII proteins as apoplastic and chitin-binding effectors, contributing to fungal virulence.
- The evolutionary analysis sheds light on the diversification of these proteins, offering valuable information for future research on fungal-plant interactions.
