Interferon
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FunctionInterferons were the first cytokines discovered and were identified by Isaacs and Lindenmann. These proteins were classified as interferons because they interfered with virus growth.[1] The initial experiments performed poorly characterized the interferons, and was based merely on bioactivity. Advances in scientific instrumentation and technique have allowed for greater understanding and visualization of not only the structure but also the mechanisms of the various types of inteferons.[2] The interferons were originally classified as leukocyte (interferon-α), fibroblast (interferon-β), and immune (interferon-γ), although today they are classified into types I (α, β, ε, κ, ω), II (γ), and III (λ).[1][2]
Type IType I interferons are homologous helical cytokines that effect a wide variety of cells pleiotropically. These effects range from antiviral activity to antibacterial, antiprozoal, immunodulatory, and cell growth regulatory functions. Without Type I interferons, the survival of the higher vertebrates would be impossible. Because of their strong antiviral and antiproliferative effects, these interferons are used in the treatment of numerous cancers, hepatitis C, and multiple sclerosis. See Multiple sclerosis. All type I interferons bind to a cell surface receptor consisting of two subunits: IFNAR1 and IFNAR2. These receptors belong to a class II helical cytokine receptor family (HCRII). Other members of this family include the interferon-γ receptor (IFNGR), tissue factor (TF), the interleukin 10 receptor (IL20R1 and IL20R2), IL-28BP, IFNLR, and IL28Rα.[4]. See more details in IntronA (Interferon alpha 2b) Interferon-αInterferon alpha has many identifiable regions with two disulfide bonds: one between the N-terminus and Helix E, and the other between Loop AB and Helix G. It has seven alpha helices and has several loop regions. The helices A, C, and F run parallel to one another, and anti-parallel to B, E, and G which run parallel to each other. Helix D does not run parallel or anti-parallel to either set, but rather runs at a 45-90 degree angle to them. Helix A consists of residues 10-12; Helix B of 40-43; Helix C of 53-68; Helix D of 70-75; Helix E of 78-100; Helix F of 109-132; and Helix G of 137-158. Interferon-βA protein growth factor that stimulates an antiviral defense interferon-beta is one of the only two known vertebrate structural genes that lacks introns.[5] Interferon-β has a 31% sequence homology to interferon-α . It is a relatively simple biological response modifier, with several identifiable regions. It consists of five alpha helices, as compared to the seven of interferon-α, as well as multiple interconnecting loop regions. Helices A, B and D run parallel to one another, and helices C and E run anti-parallel to the other three helices, but parallel to one another. Helix A consists of residues 6-23; Helix B consists of residues 49-65; Helix C consists of residues 77-91; Helix D consists of residues 112-131; and Helix E consists of residues 135-155.[6][7]. Interferon-β is used as a treatment for Multiple sclerosis, an autoimmune disease defined by Nylander and Hafler as "a multifocal demyelinating disease with progressive neurodegeneration caused by an autoimmune response to self-antigens in a genetically susceptible individual."[8] Inflammation is the primary cause of damage in MS, and though the effects of the disease are well known and various treatments exist for the disease, the exact identity of an antigen or infectious agent that causes the initiation of a myriad of symptoms is unknown.[9]. For more details see User:Chengfeng Ren/IFN beta 1a. Comparison of three interferons Signaling and Receptor InteractionsThe signaling pathways of interferons are interesting as type I interferons share the same receptors IFNAR1 and IFNAR2. Type II interferon-γ has receptors IFNGR1 and IFNGR2, but needs two interferon-γ to signal, as illustrated in the image to the right. Interestingly enough, types I and III act together in the JAK-STAT pathway, while type II acts alone. Interferon-α and -β bind to the same receptors as one another, the affinities with which they bind to IFNAR1 and IFNAR2 differ. While the binding to IFNAR2 is stronger for both in comparison to IFNAR1, interferon-β has a much stronger affinity for IFNAR1 than interferon-α.[10] Interferon-α binds to an interferon receptor mainly with helices C and G. There are many residues within 4 angstroms of one another. These residues could form many different types of bonds, illustrated in white dotted lines. Given that interferon-α does not undergo many structural changes upon binding to interferon receptor II, Quadt-Akabayov et al. have concluded that the binding mechanism is similar to that of a lock and key. Interferons -α and -β interact with a receptor at the cell surface.[11] This receptor has three domains: an N-domain, with two disulfide bonds, a C-domain, with one disulfide bond, and a linker region. The termini regions of the receptor have no secondary structure, allowing for some serious flexibility, leading to eight clashes amongst the domains.[12] Structural linkage between ligand discrimination and receptor activation by type I interferons [13]IntroductionIFNs were the first cytokines discovered more than half a century ago as agents that interfere with viral infection. Since then, IFNs have been established as pleiotropic, multifunctional proteins in the early immune response, exhibiting pronounced antiproliferative effects on cells, in addition to their strong immunomodulatory and antiviral activities. Due to their potency and diverse biological activities, IFNs are used for the treatment of several human diseases, including hepatitis C, multiple sclerosis and certain types of cancer. All type I IFNs initiate signaling by binding to the same cell surface receptor composed of two subunits called IFNAR1 and IFNAR2. The intracellular domains (ICDs) of IFNAR1 and IFNAR2 are associated with the Janus kinases (Jaks) Tyk2 and Jak1, respectively. Upon ligand binding by the IFNAR chains and formation of the signaling complex, these tyrosine kinases trans-phosphorylate and thereby activate each other. Subsequently, the activated Jaks phosphorylate STAT transcription factors, which translocate into the nucleus and activate the expression of hundreds of IFN-stimulated genes. To gain insight into how type I IFNs engage their receptor chains, how the receptor system is able to recognize the large number of different ligands, and how different IFN ligands can evoke different physiological activities, we determined the crystal structures of unliganded IFNAR1 (SD1-SD3: sub-domains 1-3), the binary complex between IFNa2 and IFNAR2, and the ternary ligand-receptor complexes of IFNa2 and IFNw binding both receptor chains. A final theoretical ternary structure including the membrane-proximal sub-domain (SD4) of IFNAR1 was also created. These structures, in conjunction with biochemical and cellular experiments, reveal that the type I IFN receptor uses a mode of ligand interaction that is unique among cytokine receptors, but conserved between different IFNs. Furthermore, ligand discrimination occurs through distinct energetics of shared receptor contacts, and differential IFN signaling is mediated by specific ligand-receptor interface chemistries that lead to different ternary complex stabilities. Interactions Between IFNAR & IFNA superposition of the two ternary complexes reveals that they have very similar overall architectures, despite the different physiological activities of the IFN ligands. This suggests that the activity differences are not due to different signaling complex architectures. The functional differences are rather mediated by specific interface chemistries that form the basis for different ternary complex stabilities. IFNAR2-IFN interaction Interferon interacts primarily with the D1 domain of IFNAR2. Arg33(IFN) appears to be the single most important residue for the interaction of the IFN ligand with IFNAR2. It forms an extensive hydrogen-bonding network with the main chain carbonyl oxygen atoms of Ile45(IFNAR2) and Glu50(IFNAR2) and the side chain of Thr44(IFNAR2). This residue is present in IFNa, IFNw, IFNb and IFNe. Two hydrophobic interaction clusters are part of the IFNa-IFNAR2 interface: the first one is formed between Leu15 and Met16 of the IFN molecule and Trp100 and Ile103 of IFNAR2; the second one comprises Leu26, Phe27, Leu30 and Val142 of the ligand and Met46, Leu52, Val80 and the methyl group of Thr44 of the receptor. Replacing Leu30(IFN) with alanine reduces affinity by three orders of magnitude (the second most important residue for binding). This is surprising, as it is not engaged in any intimate contacts with IFNAR2 residues. One reason for its importance might be a stabilizing effect on the position of Arg33(IFN). Most of the residues involved in the IFNa2-IFNAR2 interaction are also found in the IFNw-IFNAR2 interface of the IFNw ternary complex.  A significant difference in the IFNAR2 interface between IFNa2 and IFNw is related to Arg149 in IFNa2, which is replaced with Lys152 in IFNw. In the IFNw-IFNAR2 interface, this residue forms an intramolecular salt bridge with Glu149(IFN), but does not contact Glu77 of the receptor. IFNAR1-IFN interactionBecause of the lower resolution of the IFNa ternary complex, we focused on the IFNw complex in our analysis of the IFN-IFNAR1 interface. In the IFNw-IFNAR1 complex, the ligand-binding site of IFNAR1 only contains two hotspot residues we could experimentally confirm, Tyr70(IFNAR1) and Phe238(IFNAR1). Substituting these residues by alanine reduces the affinity to all tested IFN ligands by more than 10-fold. On IFNw, mutation studies have shown that a charge-reversal mutation of Arg123 (Arg 120 on IFNa) leads to a total loss of activity.  Indeed, this residue forms a salt bridge with Asp132(IFNAR1) in addition to a hydrogen bond with Ser182(IFNAR1). Substitution of glutamate for Arg123(IFN) would lead to electrostatic repulsion with Asp132(IFNAR1). The low affinity of IFNAR1 for the ligand appears to be functionally relevant, as weak binding to IFNAR1 is conserved between all alpha IFNs. Three amino acid substitutions on IFNa2 at positions His57, Glu58 and Ser61 to alanine or to Tyr, Asn, and Ser, respectively, confer tighter binding to IFNAR1, but leave the affinity to IFNAR2 essentially unaltered. Implications for the binding mode of IFNbIFNb exhibits 30% and 33% sequence identity with IFNw and IFNa2, respectively. Superimposing human IFNb onto IFNw in our ternary complex structure leads to only two clashes of side chains (Tyr92 and Tyr155) with the receptors, indicating that the IFNb ligand could be easily accommodated by the receptors in a position similar to IFNw and IFNa2. Furthermore, the superposition of IFNb onto IFNa2 in complex with IFNAR2 shows that Trp22 in IFNb and Ala19 in IFNa2 overlay onto each other. As a result, Ala19(IFN), when mutated to tryptophan, promotes an increased binding affinity to IFNAR2, which is a result of the contact made to Trp100 in IFNAR2 (as shown by double mutant cycle analysis).
Structural MovementsStructural pertubations upon bindingOne of the more controversial aspects of cytokine signaling is whether receptor binding is sufficient to generate activity, or if it has to be accompanied by structural perturbations. The type I interferon signaling complex is a rare example of a cytokine receptor complex were the structures of all the components making up the biologically active complex were determined to high resolution in both their free and bound forms. A comparison of the unbound NMR structure with the ternary complex structure of interferon shows a small expansion during complex formation. Both IFNAR1 and IFNAR2, however, undergo significant domain movements upon binding. Using the D1 domain as anchor, a clear outwards movement of the D2 domain of IFNAR2 upon binding, on a scale of 6-12 Å, is observed (comparison of the unbound receptor (1n6u) with the binary IFNa2-IFNAR2 complex). The superimposition of the IFNa2-IFNAR2 binary complex with IFN-IFNAR2 in the ternary complexes reveals an additional domain movement of 6-9 Å, and even between the ternary IFNa and IFNw complexes a movement of 3-5 Å is observed. The D2 domain is engaged in crystal contacts in all three structures, and it remains an open question if the conformational changes in IFNAR2 are physiologically relevant. Still, these movements could change the proximity or orientation of the ICDs and associated Jaks within the cell. The low-affinity receptor chain, IFNAR1, also undergoes major conformational changes upon ligand binding. When using D1 as anchor, D3 is moving inwards (toward the ligand) by ~15 Å. This would generate an even larger movement of the membrane-proximal SD4 domain and the transmembrane helix. The conformational changes in IFNAR1 are necessary to form the full spectrum of interactions with the IFN ligand, and to form a stable signaling complex that is able to instigate downstream signaling. In contrast to SD3, SD4 seems to be highly flexible (even more than D2 of IFNAR2). One might suggest that the conformational changes in IFNAR1 by itself will be responsible for a reduced binding affinity of IFNAR1 and may slow down the rate of ligand association to IFNAR1 directly from solution. See Also
3D Structures of interferon
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References
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