Molecular characterization and heterodimerization of three type I interferon receptor subunits (CaCRFB1, CaCRFB2, CaCRFB5) in the humpback grouper (Cromileptes altivelis).

Abstract

Type I interferons (IFNs) are a class of pleiotropic cytokines that play a pivotal role in host defense against pathogenic invaders by binding with specific heterodimeric receptors. Among teleosts, these receptors consist of diverse subunits, including CRFB1, CRFB2, and CRFB5, whereas their pairing patterns and structural basis of interactions remain ill-defined. In this study, we identified and characterized three type I IFN receptor genes (CaCRFB1, CaCRFB2, and CaCRFB5) in the humpback grouper (Cromileptes altivelis), all sharing a common architecture of two extracellular FNIII domains. Furthermore, all three CaCRFB genes exhibited not only constitutive expression across all examined tissues but also significant transcriptional upregulation in response to poly I:C, LPS and pathogenic challenges (Vibrio harveyi and nervous necrosis virus), highlighting their active involvement in innate immunity. To elucidate their assembly mechanisms, we integrated molecular docking with co-immunoprecipitation (Co-IP) assays targeting their extracellular domains. Our results validated the canonical heterodimerization of the short-chain CaCRFB5 with both long-chain subunits (CaCRFB1 and CaCRFB2) as well as a noncanonical interaction of CaCRFB1/CaCRFB2, suggesting a more intricate receptor pairing in teleosts. Structural interface analysis revealed that these interactions are principally driven by electrostatic forces, including hydrogen bonds and salt bridges. Intriguingly, while the membrane-proximal FNIII domain II mediates canonical interactions (CaCRFB1/CaCRFB5 and CaCRFB2/CaCRFB5), it is the membrane-distal FNIII domain I that orchestrates the noncanonical CaCRFB1/CaCRFB2 pairing. These findings shed light on the structural basis of receptor heterodimerization, enriching the understanding of the teleost type I IFN system.