Abstract 2685 Structural studies of the IFNλ4 receptor complex using cryoEM enabled by protein engineering

IFNL4 (Interferon Lambda 4) is a recently discovered type III interferon that plays a critical role in the immune response, particularly in the context of viral infections.Its genetic variation has been shown to significantly affect an individual's susceptibility to viral infections, particularly hepatitis C virus (HCV), as well as responses to other pathogens.While IFNL4's role in antiviral immunity is still under investigation, it is emerging as an important factor in the pathogenesis of viral diseases, particularly chronic infections.Furthermore, its potential implications for personalized medicine, especially in antiviral therapy, are a focus of ongoing research, with the goal of improving therapeutic outcomes for individuals with different IFNL4 genotypes.While predicted to be structurally similar to IFNl1-3, IFNl4 shares only 28% amino acid sequence identity with its closest relative IFNl3 and has a distinct biochemical composition resulting in IFNl4 being one of the most positively-charged known cytokines.These differences evoke intriguing structural questions regarding potential differences in receptor engagement between IFNl3 and IFNl4 and their resulting influences on cell signaling and IFN function.We employ protein engineering to address the affinity challenges for structural determination of the ternary IFNl cytokine-receptor complex.Using cryogenic electron microscopy (cryoEM), we resolve the 72 kDa structures of IFNl4 and IFNl3 in complex with IFNlR1 and an affinitymatured IL10Rb in high resolution.Structural analysis reveals key insights into the extracellular binding mechanism of IFNl4 to its receptors, revealing distinct differences in residue interactions and receptor geometry compared to IFNl3, which provide potential mechanistic explanations for differences in the in vitro function of IFNl3 and IFNl4.Our results also highlight the continued importance of experimentally determining structures of protein complexes, as our attempts to model the structure of the IFNl4-IFNlR1-IL10Rb complex using computational tools for structure prediction did not capture this difference in receptor geometry.