Molecular analysis of the type III interferon complex and its applications in protein design and engineering

Type III interferons (IFNL1-4) are proteins with critical roles in the immune system and signal only at barrier tissues, making them attractive therapeutic candidates. However, structural studies of the proteins and their signaling complexes have been limited by low-affinity protein-protein interactions and challenging expression conditions in the lab. While previous efforts have utilized extensive protein engineering to overcome these barriers, we use a combination of computational and experimental techniques to generate a detailed molecular analysis of the type III IFNs. Using molecular modeling and simulations, we quantify differences in residue contact and strain fluctuation, produce detailed free-energy landscapes, and reveal previously unknown structural features of the engineered and wild type extracellular signaling complexes; we then leverage this information to guide engineering at the receptor-receptor interface, increasing complex formation by as much as 2.5-fold and identifying hot spots of interactions between the ligands and receptors. This two-pronged approach to protein design and engineering enables analysis of inaccessible protein complexes to generate molecular insights into IFNL behavior, elevating approaches to harness their therapeutic potential.