Small Disulfide Proteins with Antifungal Impact: NMR Experimental Structures as Compared to Models of Alphafold Versions

In response to the growing challenge of emerging resistance to conventional antifungal drugs, antifungal proteins (AFPs) of filamentous Ascomycetes origin such as PAF, PAFB, and AFPg, have been discovered in recent years. Understanding the structure of AFPs is crucial for elucidating their antifungal mechanisms and provides a theoretical foundation for the design of new therapeutic agents. While nuclear magnetic resonance (NMR) has proven effective in determining the structures of small proteins, some AFPs structures remain unresolved, necessitating the use of alternative structural prediction methods. Through bioinformatics analysis and heatmaps of amino acid sequence identity and similarity matrix, we categorized AFPs into three major classes and six subcategories, revealing structural and bioactivity differences. We employed AlphaFold (AF) to predict the 3D structures of six different AFPs and compared these predictions with NMR-derived structures to assess the accuracy of AF predictions. The results demonstrated a high degree of consistency between AF and NMR structures, with AF excelling in structural quality assessment and accurately capturing complex disulfide bond patterns. Further analysis revealed that both AF2 and AF3 models outperform the NMR model in overall structural quality and coherence, with AF3 showing the best performance. This study verifies the reliability of AF in the structural prediction of cysteine-rich AFPs, offering important support for the rational design of new protein-based antifungal drugs.