Altered Stereostructures of the DNA-Binding Domains of Variant Mating Proteins of Ophiocordyceps sinensis and the Wild Insect–Fungal Complex

The MATα_HMGbox and HMG-box_ROX1-like domains of the MAT1-1-1 and MAT1-2-1 proteins, respectively, play essential roles in DNA binding and the subsequent regulation of gene transcription, controlling <i>Ophiocordyceps sinensis</i> sexual reproduction. Alternative splicing, differential occurrence and transcription of the <i>MAT1-1-1</i> and <i>MAT1-2-1</i> genes have been demonstrated in <i>Hirsutella sinensis</i> (GC-biased Genotype #1 of the 17 <i>O. sinensis</i> genotypes), suggesting self-sterility under heterothallic or hybrid outcrossing. In this study, the MATα_HMGbox domains of MAT1-1-1 proteins in wild-type <i>Cordyceps sinensis</i> isolates were shown to cluster into 5 clades in the Bayesian clustering tree and belong to diverse stereostructure morphs under 19 AlphaFold codes. The HMG-box_ROX1-like domains of MAT1-2-1 proteins, on the other hand, were shown to cluster into 2 branched Bayesian clades and belong to stereostructure morphs under 25 AlphaFold codes. Correlation analysis revealed that 1-3 amino acid substitutions in the DNA-binding domains of the mating proteins resulted in altered hydrophobicity and secondary and tertiary structures of the DNA-binding domains of the proteins, especially altered stereostructures of the hydrophobic cores formed by 3 critical α- helices within the functional domains of the proteins. Fungal origin analysis revealed possible heterospecific fungal sources of mating proteins with stereostructure variations in wild-type <i>C. sinensis</i> isolates, suggesting that alterations in DNA binding function and the subsequent regulation of mating-related gene transcription are involved in ensuring the accuracy and genetic diversity of heterothallic and hybrid reproduction of <i>O. sinensis</i> during the lifecycle of the <i>C. sinensis</i> insect-fungal complex.