Type I interferons were discovered over 50 years ago as antiviral agents. Subsequent research has shown that the many IFN sub-types show differential activities through common receptor chains. Our studies show that the overall architectures of receptor binding to both IFNα2 and IFNω are nearly identical (), and that the answer to how different IFNs are capable of inducing differential functional effects appears to result from ligand discrimination through distinct receptor binding chemistries, which dictate the respective stabilities of the receptor-ligand interactions. The distinct binding chemistries are achieved primarily by differential energetics of shared anchor points, and to a lesser extent by key amino acid substitutions between IFNs. These ligand-specific differences in the extracellular complex stabilities manifest as perturbations in downstream signaling cascades, in both linear and non-linear fashions. Mechanistically, different complex stability kinetics could control the relative Jak/Tyk activity towards intracellular substrates of greater or lesser accessibility, which would, in turn lead to distinct downstream effector activation profiles and ultimately impact induction of IFN-responsive genes. In this respect, recognition-mediated tuning of differential signaling by the Type I IFN receptor system is quite unique for a transmembrane receptor, but has parallels to the antigen ‘proofreading’ ability of the T cell receptor to differentially respond to self and foreign peptide-MHC molecules presenting subtly different peptide recognition chemistries.
In the context of prior cytokine receptor structures, IFNAR1 is particularly striking, with participation of three subdomains and a conformational change upon IFN binding ( and S3
). That this is a bona-fide ligand-induced conformational change is corroborated by the importance of the SD1 domain for ligand binding, and by FRET measurements suggesting conformational changes in the ectodomain of IFNAR1 upon IFN binding (Strunk et al., 2008
). The conformational change in IFNAR1 is required to form the full spectrum of interactions with the ligand and to allow the formation of a ternary complex that is stable enough to facilitate trans-phosphorylation between Jak1 and Tyk2. Thus, ligand binding to IFNAR1 will be accompanied by an energetic cost associated with the structural rearrangements required to bring a key hotspot residue into contact, and could play a role in ‘tuning’ responsiveness to different IFN ligands. We suggest that the required conformational change contributes to the reduced binding affinity of IFNAR1 and may result in tighter control of IFN signaling.
In addition to the conformational change, the role of IFNAR1 in ligand responsiveness is also unique compared to IFNAR2. IFNAR1 is not optimized for high binding affinity, but rather for functional plasticity. That is, in contrast to the interaction with IFNAR2, binding energy is distributed over a large number of amino acid contacts with relatively low individual contributions and with much lower cooperativity, altogether resulting in lower affinity. For early STAT activation, which is required for the antiviral cellular response, transient ligand interaction with IFNAR1 appears to be advantageous (Moraga et al., 2009
). High stability of the ternary complex seems to be more important for a subset of IFN activities requiring prolonged activation of IFN signaling pathways (Coelho et al., 2005
; Jaitin et al., 2006
). The relatively large binding interface of IFNAR1 for IFN involving 3 FNIII-like domains provides a versatile means for fine-tuning the binding affinity towards different IFNs and tailoring differential response patterns.
The molecular basis of IFNAR cross-reactivity is unique compared with other shared receptor systems, such as gp130 and common gamma chain (γc
), and this likely reflects the fact that the IFN interaction chemistry controls signal initiation. Gp130 engages different cytokines through entirely distinct binding surfaces that do not appear to share anchor points, whereas γc
engages in degenerate binding largely through shape complementarity (Wang et al., 2009
). What sets the IFNAR system apart is that the IFNAR1/2 heterodimer recognizes and transduces the signal for all 16 IFN sub-types, whereas in the other shared cytokine receptors, signal specificity is determined by different ligand-specific co-receptors hetero-dimerizing with the shared receptor. In this way, the recognition chemistry of gp130 and γc
are not important arbiters of signaling specificity.
With regards to function, our mutational and substitution experiments suggest a model whereby ablating or swapping key IFN-specific residues that engage in receptor interactions narrows the functional distinction between IFNs. Importantly, however, the mutational analysis also shows that the local environment of these contacts plays an important role in determining their energetic values in the respective IFN complexes. Mutation of individual positions has complicated energetic consequence. Therefore ligand-specific residues are not “plug-and-play” in a manner that easily allows one to recapitulate IFN sub-type behavior by point mutagenesis. This is to be expected given that the functional distinction of IFN ligands arose, in part, through co-evolution of broad receptor-ligand interaction surfaces over hundreds of millions of years. A surprising exception to this was the K152R gain of function mutation in IFNω, which, clearly, is a highly modular contact point.
Ligand-specific differences in the stabilities of the complexes are also reflected in variances in the kinetics of receptor downregulation, which terminates signaling. Our studies revealed that increased binding affinities towards IFNAR1 (IFNα2(YNS) mutant) or IFNAR2 (IFNω(K152R) mutant) strongly enhance receptor downregulation, which very likely explains a much more rapid decline in p-STAT activation compared to IFNα2(wt) and IFNω(wt). Increased IFNAR2 downregulation by the higher affinity IFNβ, compared to IFNα2, has been previously suggested to be responsible for differential cellular responses (Jaitin et al., 2006
; Kalie et al., 2007
). Here, we have designed an IFN mutant with increased binding affinity towards IFNAR2, which surprisingly induces even stronger downregulation of IFNAR2. Increased IFNAR2 downregulation could explain why the substantially increased binding affinity of these IFN mutants is not accompanied by a significant increase in their AV potency, because it is very likely responsible for a rapid decrease in p-STAT levels, as seen after stimulation with IFNα2(YNS) and IFNω(K152R).
In contrast to AV activity, which requires only very low doses of IFN to reach saturation, AP activity benefits from an increased binding affinity (Kalie et al., 2008
). Cells need to sense very low levels of IFN and act very fast in order to clear viral infections in their initial stages. On the other hand, antiproliferative activity, which is often linked with apoptosis and tissue damage, needs to be under tighter control to prevent unnecessary damage. These activities will therefore be more tunable over a broad range to changes in the kinetics and strength of the downstream signaling. IFNs, by forming a gradient of complex stabilities will induce specific profiles of signal activation that will lead to diverse antiproliferative potencies. Taken together, differential IFN signaling activities are mediated by both non-linear signaling and non-linear receptor desensitization mechanisms. This type of “ligand proofreading” provides a mechanistic model, now together with a structural framework, for how a common receptor can respond in a graded fashion to different ligands.