Heteromorphic tertiary structures of MAT1-1-1 and MAT1-2-1 protein variants simultaneously produced in pairs by Ophiocordyceps sinensis strains

The MAT_HMGbox domain in the MAT1-1-1 protein and the HMG-box_ROX1-like domain in the MAT1-2-1 protein play essential roles in DNA binding and regulating the transcription of genes that control sexual reproduction in Ophiocordyceps sinensis. Previous studies have reported differential occurrences, differential transcription, and alternative splicing of the MAT1-1-1, MAT1-2-1, and pheromone receptor genes in Hirsutella sinensis (Genotype #1 among 17 genome-independent genotypes of O. sinensis fungi). In this study, we further demonstrated that the DNA-binding domains of the paired MAT1-1-1 and MAT1-2-1 proteins derived from each of the 20 purportedly "pure" O. sinensis strains exhibit heteromorphic tertiary structures, as predicted by AlphaFold 3D structural modeling. These differentially paired mating proteins are characterized by different truncations, diverse 1-4 amino acid substitutions at distinct sites, altered hydrophobicity and secondary structures, and heteromorphic tertiary structures, indicating divergency in the fungal origins of the mating proteins within the mycologically and genetically impure O. sinensis strains. Our findings support the self-sterility hypothesis for O. sinensis and likely ensure the fidelity and genetic diversity of heterothallic or hybrid reproduction during the lifecycle of the Cordyceps sinensis insect-fungal complex.