Designing monomeric IFNγ: the significance of domain-swapped dimer structure in IFNγ immune responses

IFN{gamma} can initiate immune responses by inducing the expression of major histocompatibility complex molecules, suggesting its potential for cancer immunotherapy. However, it also has an immunosuppressive function that limits its application as a therapeutic agent. IFN{gamma} has a characteristic domain-swapped dimer structure with two of the six -helices exchanged with each other. As we hypothesized that the contrasting functions of IFN{gamma} could be attributed to its unique domain-swapped structure, we designed monomeric IFN{gamma} by transforming the domain-swapped dimer structure of wild-type IFN{gamma}. We conjectured the evolution of this domain-swapped dimer and hypothesized that the current IFN{gamma} structure emerged through shortening of the loop structure at the base of the swapped domain and the accumulation of hydrophobic amino acids at the newly generated interface during domain-swapping. We then designed and generated a stable monomeric IFN{gamma} by retracing this evolutionary process, complementing the lost loop structure with a linker and replacing the accumulated hydrophobic amino acids with hydrophilic ones. We determined that the designed variant was a monomer based on molecular size and number of epitopes and exhibited activity in cell-based assays. Notably, the monomeric IFN{gamma} showed a qualitatively similar balance between immunostimulatory and immunosuppressive gene expression as wild-type IFN{gamma}. This study demonstrates that the structural format of IFN{gamma} affects the strength of its activity rather than regulating the fate of downstream gene expression.