Edited by Luke O'NeillThe interaction of IFN-␤ with its receptor IFNAR1 (interferon ␣/␤ receptor subunit 1) is vital for host-protective anti-viral and anti-proliferative responses, but signaling via this interaction can be detrimental if dysregulated. Whereas it is established that IFNAR1 is an essential component of the IFNAR signaling complex, the key residues underpinning the IFN-␤-IFNAR1 interaction are unknown. Guided by the crystal structure of the IFN-␤-IFNAR1 complex, we used truncation variants and site-directed mutagenesis to investigate domains and residues enabling complexation of IFN-␤ to IFNAR1. We have identified an interface on IFNAR1-subdomain-3 that is differentially utilized by IFN-␤ and IFN-␣ for signal transduction. We used surface plasmon resonance and cell-based assays to investigate this important IFN-␤ binding interface that is centered on IFNAR1 residues Tyr 240 and Tyr The type I IFNs, including 14 IFN-␣ and lone IFN-␤, IFN-⑀, and IFN-, have critical roles in response to viral and bacterial infections and cancers (1, 2). They are also applied clinically for the treatment of hepatitis virus B and C (1), cancers including melanoma (3), and multiple sclerosis (4). Although they show clinical efficacy, their use is restricted by dose-limiting toxicities, including leukopenia, nausea, fatigue, neurological disorders (3), and localized cutaneous effects (5). All type I IFNs engage their cognate receptors, IFNAR1 and IFNAR2, to activate the canonical JAK-STAT signaling pathway, but ligand engagement can also activate alternative signaling pathways (6). Despite sharing these receptor components, there are IFN subtype-specific elements to signaling; compared with IFN-␣, IFN-␤ has specific roles in osteoclastogenesis (7), control of chronic viral infection (8), the potent induction of apoptotic pathways required for control of tumor cell growth, and the development of B cells and myelopoiesis (9).Structural insight into the IFN-IFNAR 5 interactions has been gleaned from the crystal structures of a human IFN-␣2 variant and human IFN-in complex with both IFNAR1 and IFNAR2 (10). Furthermore, specific insight into the mode of IFN-␤-mediated activation of IFNAR1 was obtained from the crystal structure of the murine IFN-␤-IFNAR1 complex (11). Comparison of these structures and evidence from the literature (12) suggests that the minimal ligand binding domains for human and mouse IFNAR1 are similar and sit broadly across the three membrane distal SDs (SD1-3) of the receptor with limited involvement of the membrane proximal subdomain, SD4 (Fig. 1A). It has also been shown that key residues discriminate between ligands and that there is potential for ligand-specific interaction interfaces (10, 11).