type i interferon structures: possible scaffolds for the interferon-alpha receptor complex

Type I interferon structures: Possible scaffoldsfor the interferon-alpha receptor complex

Tattanahalli L. Nagabhushan, Paul Reichert, Mark R. Walter, andNicholas J. Murgolo

Abstract: The structures of several type I interferons (IFNs) are known. We review the structural information knownfor IFN alphas and compare them to other interferons and cytokines. We also review the structural information knownor proposed for IFN–cell receptor complexes. However, the structure of the IFN – cell receptor – IFN receptor2(IFNAR2) and IFN receptor1 (IFNAR1) complex has not yet been determined. This paper describes a structural modelof human IFN-IFNAR2/IFNAR1 complex using human IFN-�2b dimer as the ligand. Both the structures of recombi-nant human IFN-� 2b and IFN-� were determined by X-ray crystallography as zinc-mediated dimers. Our proposedmodel was generated using human IFN-�2b dimer docked with IFNAR2/IFNAR1. We compare our model with the re-ceptor complex models proposed for IFN-� and IFN-� to contrast similarities and differences. The mutual binding sitesof human IFN-�2b and IFNAR2/IFNAR1 complex are consistent with available mutagenesis studies.

Key words: three dimensional structure, antiviral activity, receptor, interferon.

Résumé : On connaît les structures de plusieurs interférons (IFN) de type I. On passe en revue les informationsstructurales connues pour les IFN alpha et on les compare à celles connues pour d’autres interférons et cytokines. Onpasse aussi en revue les informations structurales connues ou proposées pour les complexes IFN–cellule réceptrice.Toutefois, les structures du complexe INF – cellule réceptrice – IFN récepteur 2 (IFNAR2) et IFN récepteur 1 (IFNAR 1)n’a pas encore été déterminée. Dans ce travail, utilisant le dimère interféron humain-�2b comme ligand, on décrit unmodèle structural du complexe humain IFN-IFNAR2/IFNAR1. Faisant appel à la diffraction des rayons X sur desdimères reliés au zinc, on a déterminé les structures des recombinants humains humain-�2b ainsi que IFN-�. Notremodèle proposé a été généré en utilisant le dimère humain IFN-�2b arrimé au IFNAR2/IFNAR1. On a comparé notremodèle avec les modèles du complexe récepteur proposés pour IFN-� et IFN-� pour mettre en relief les similarités etles différences. Les sites de fixation mutuels du dimère humain IFN-�2b et du IFNAR2/IFNAR1 sont en accord avecles études disponibles de mutagénèse.

Mots clés : structure tridimensionnelle, activité antivirale, récepteur, interféron.

[Traduit par la Rédaction] Nagabhushan et al. 1173

Introduction

GeneralThe diverse and multiple biological properties of

interferons (IFNs) have driven the need for an understandingof their activity at the molecular level. In 1957, Issacs andLindenmann (1) discovered IFNs as antiviral agents. IFNsare a family of structurally and functionally related proteinsthat exhibit antiviral and antiproliferative effects on variouscell types. They have been shown to exhibit various potentimmunomodulatory effects including regulation of naturalkiller cell activity and modulation of major histocompatability

antigen expression as well as antiproliferative activityagainst malignant cells (1–5).

Classification of interferonsIFNs were historically classified according to the cells of

origin (i.e., leukocyte, fibroblast, and immune IFN). Moremodern designations are based on immunogenic propertiesand gene sequences. The major classes of IFNs are IFN-�, -� ,-�, and -�, which are also designated type I (acid stable),and IFN-� designated type II (acid labile). The IFN-� genefamily is known to consist of at least 14 distinct humanmembers that encode 12 different proteins (6). Sequencing

Can. J. Chem. 80: 1166–1173 (2002) DOI: 10.1139/V02-158 © 2002 NRC Canada

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Received 14 December 2001. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 30 August 2002.

Dedicated to the memory of Professor Raymond U. Lemieux.

T.L. Nagabhushan.1 Schering Plough Research Institute, Discovery Research, Kenilworth, NJ 07033, U.S.A.P. Reichert. Schering-Plough Research Institute, Department of Structural Chemistry, Kenilworth, NJ 07033, U.S.A.M.R. Walter. Center for Biophysical Sciences and Engineering, Department of Microbiology, University of Alabama atBirmingham, Birmingham, AL 35294, U.S.A.N.J. Murgolo.1 Schering-Plough Research Institute, Department of Bioinformatics, Kenilworth, NJ 07033, U.S.A.

1Corresponding authors (e-mail for T.L.N.: [email protected]; e-mail for N.J.M.: [email protected]).

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of mammalian genomes has led to identification of addi-tional classes including limitin (7) and IFN-� (8). A numberof synthetic and hybrid genes have been expressed to pro-duce unnatural proteins including consensus IFN (9). Con-sensus IFN is a synthetic IFN-� designed by assemblingeach of the most frequently observed amino acids in severalIFN-� subtypes (9). The human IFN-� species are particu-larly homologous in amino acid sequence (about 78%) andgenerally display a high level of species specificity in theirbiological activities. Primary amino acid comparisons of theIFN-� subtypes with human IFN-� and IFN-� reveal se-quence identities of about 30% and 10%, respectively, (10).The type I IFN-� family contains proteins of 166 amino acidresidues except for IFN-�2, which has 165 residues, andIFN-� with 172 residues. All the IFN-� subtypes containtwo disulfide bridges with Cys1–Cys99 and Cys29–Cys138,except IFN-�2, which lacks position 44 and forms thedisulfide bridges with Cys1–98 and Cys29–Cys138 and forIFN-�8, which forms the first disulfide bridge with Cys1–Cys100. IFN-� has only one disulfide bridge at Cys31–Cys141(11). The most biophysical information has been reportedfor recombinant E. coli derived human �- and �-interferon.The homogeneity of human recombinant IFN-�2b has beenanalyzed using various physio–chemical methods developedto analyze the quality of interferon preparations includinggel electrophoresis, reverse phase HPLC, NMR, variousmass spectral methods, and circular dichroism (12).

IFN-� and IFN-� interact with two receptor componentson cell surfaces, interferon alpha receptor 1 (IFNAR1) andinterferon alpha receptor 2 (IFNAR2), which belong to theclass II cytokine receptor superfamily (13). Both subunits ofthe receptor are required for selective ligand recognition andthe receptors recognize distinct ligand epitopes (14).IFNAR1 contains four extracellular Ig-like domains, whileIFNAR2 contains two extracellular Ig-like domains. Se-quence similarity to tissue factor and IFN-� receptors (bothof which contain two Ig-like extracellular domains) suggestIFNAR1 and IFNAR2 will adopt similar folds. The avail-ability of high-resolution structures of these receptors has fa-cilitated homology modeling of IFNAR1 and IFNAR2. Suchmodels are useful for mapping ligand and antibody epitopes.

Structural information on IFN-�, -�, and -� andinterleukin-10

IFN-�Although crystallization of human recombinant IFN-�2a

was first reported in 1982 (15), high quality crystals of thezinc-mediated non-covalent dimer of human recombinantIFN-�2b were not reported until 1996 (16). The crystallo-graphic asymmetric unit contains three non-covalent dimersof the IFN-�2b monomer shown in complimentary orienta-tions in Fig. 1. The zinc-binding site is formed from adja-cent glutamic acid residues (Glu41 and Glu42) located onthe 310 helix at the C-terminal end of the AB loop of eachmonomer. The overall fold of IFN-�2b is similar to the pre-viously determined structure of murine recombinant IFN-�(17). Each monomer of IFN-�2b is composed of five �-helices,labeled A–E, linked by one overhand connection (AB loop)and three short segments (BC, CD, and DE loops). The to-pology of IFN-�2b resembles the classical up-up-down-downfour helix bundle motif: helices A, B, C, and E comprise the

helix bundle (Fig. 1). IFN-�2b is distinguished from theclassical topology by helix D (consisting of 20 residues) thatreplaces the second extended overhand loop (CD loop) ob-served in class 1 long chain cytokines (18).

Multidimensional heteronuclear NMR spectroscopy wasused to determine the solution structure of recombinant hu-man IFN-�2a at pH 3.5 at 2–2.2 mM protein concentration(19). The lower pH was required for structure determinationbecause unspecific aggregation at higher pH led to low-quality solution 1H NMR spectra with broad resonance lines.As observed for the crystal structure of IFN-�2b, IFN-�2aconsists of five �-helices, four of which are arranged to forma left handed helix bundle with an up-up-down-down topol-ogy and two overhead connections. Among the structurallyrelated four helical bundle cytokines, the structure ofIFN-�2a is most similar to that of IFN-�2b, murine IFN-� ,and human IFN-�. The crystal structure of ovine IFN-� (20)is similar to the crystal structures of IFN-�2b, human IFN-�,and murine IFN-� and the NMR solution structure of IFN-�2a.

IFN-�To date, X-ray crystallographic structures have been deter-

mined for murine E. coli derived IFN-� (17) and CHO cellderived glycosylated human IFN-� (21). The overall topol-ogy of both murine and human recombinant IFN-�monomeric structures is most similar to IFN-�2b. Interest-ingly, human IFN-� and IFN-�2b both crystallize as zinc-mediated dimers (Fig. 2B). However, the human IFN-� zinc-mediated dimer forms a different dimer interface comparedwith the IFN-�2b crystal structure. The dimer interface ofhuman IFN-� contains a zinc atom that is coordinated byhistidine residues from adjacent monomers. A water mole-cule completes the tetrahedral zinc coordination betweenHis121 (helix D) and Glu43 (AB loop) of one monomer andGln94 (AB loop) and His97 (helix C) of the complimentarymonomer. Dimerization has been observed for other helicalcytokines and in some cases has been correlated with recep-tor activity (18). The biological significance of dimer forma-tion of human IFN-� and IFN-�2b is unclear.

IFN-�In 1991, the first human interferon structure determined

was of recombinant human E. coli derived IFN-� (22)(Fig. 2C). Human IFN-� is a homodimer in solution andcrystallizes with two dimers related by non-crystallographictwofold axis in the asymmetric unit. The protein was primarily

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Nagabhushan et al. 1167

Fig. 1. Molecular structure of IFN-�2b. (A) Ribbon diagram;(B) dimer. (A) Helices A–E are in cyan, A–B loop 310 helicesare in magenta. (B) Independent monomers are in cyan andgreen; zinc is indicated by a yellow sphere.



�-helical with six helices in each subunit. Subsequently, therecombinant rabbit IFN-� structure was reported to be topo-logically identical to human IFN-� (23). Both structureswere predominantly �-helical with extensive interdigiationof the �-helical segments of the two polypeptide chains. Thehigh resolution structure (2 Å) of bovine IFN-� reported in2000 was most similar in overall topology to the humanIFN-� (3.5 Å) (24). The structure of a biologically activesingle chain mutant of human IFN-� was reported in 2000(25). The single-chain mutant was derived by linking thetwo peptide chains of the IFN-� dimer by a seven-residuelinker and changing His111 in the first chain to an asparticacid residue.

Interleukin-10Interleukin-10 (IL-10) is a cytokine that inhibits produc-

tion of pro-inflammatory cytokines including IFN-� andTNF-�. Physiochemical studies on recombinant human IL-10 indicate that human IL-10 is predominately a non-covalent dimer at neutral pH with two disulfide bridges (26).The crystal structure of recombinant human CHO cell de-rived IL-10 is a dimer composed of identical polypeptidechains related by a twofold axis (27) (Fig. 2D). Each IL-10monomer consists of six �-helices. The main-chain folds re-semble IFN-� in which the structural integrity of each do-main is dependent on the intertwining of helices from eachpeptide chain. IL-10 and IFN-� structures have comparableoverall topology (v shape), interdomain angle (129° in bothstructures), and five amino acid sequences conserved in allamino acid sequences in the core. The high resolution crys-tal structure of refolded E. coli derived human IL-10 (28)has overall topology most similar to the recombinant CHOcell derived IL-10.

Dimer interactionsIFN-�2b, human IFN-�, IL-10, and IFN-� have dimeric

units in their crystal structures (Figs. 2A–D). Both IFN-�2b

and human IFN-� crystallized as zinc-mediated dimers;however, IL-10 and IFN-� crystallize as non-covalent inter-calated dimers. All of these cytokines bind to class 2cytokine receptors. The dimeric form of IL-10 and IFN-� areimportant for optimal receptor activation, whereas there isno evidence that IFN-� or IFN-� dimers are important foractivating their receptors.

IFN-�2b zinc-mediated dimer is most similar to humanIFN-� at the monomer level in the overall topology, folds,and elements of secondary structure. The major structuraldifferences of the main chain reside in the exposed loops.Differences include (i) helix B in IFN-�2b is kinked, whichis absent in human IFN-�; (ii) helix E in IFN-�2b is rotatedaround its axis relative to the orientation in human IFN-�;(iii) and human IFN-� is glycosylated at a single site Asn80at the end of helix C, whereas IFN-�2b is non-glycosylated.All the alpha interferons except for one species areglycosylated. Glycoslation does not interfere with dimer for-mation in human IFN-�.

IL-10 and human IFN-� display the best overall structuralidentity with each other; however, they do not share any sig-nificant sequence homology. While each domain of IL-10 re-sembles the long chain monomeric cytokines, the dimer foldof IL-10 (Fig. 2D) is most like IFN-� (Fig. 2C). The two no-table differences in the dimers are the size (length of helices)and orientation of the domains. In IL-10 the helix bundlesare perpendicular to one another, while in IFN-� the domainsare oriented at an angle of about 60°. The domains of IL-10are constrained to exhibit the 90° interdomain angle due tothe conformations of the crossover connections that areformed. Analysis of interactions in the core of IL-10 andIFN-� shows a close structural correspondence of a five resi-due cluster (IFN-� motif). IL-10 and IFN-� both containsimilar flexible regions.

Members of the type I IFN family have homology to eachother, bind to the same cell surface receptors (type II), andhave overlapping function (6, 29–31). This review is specific

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1168 Can. J. Chem. Vol. 80, 2002

Fig. 2. Comparison of dimer structures of (A) IFN-� 2b; (B) IFN-�; (C) IFN-�; and (D) IL-10. Each independent monomer is either incyan or green. For IFN-� 2b and IFN-� the coordinate zinc is indicated by a yellow sphere.



to interferon and interferon-like structures. Several broad re-views on type I and type II cytokines are available (32).

Currently, crystal structures are not available for any typeI interferon receptor complex. Here we review structural fea-tures on the related IFN-� and IL-10 receptor complexes.Based on this analysis, we propose a model for the inter-feron/interferon-receptor complex.

Comparison of the structures of IFN-� and IL-10receptor complexes

The crystal structure of IFN-� bound to its high affinityreceptor (IFN-�R1) was first completed in 1995 (33). Thecomplex contains two IFN-�R1s that bind the identical two-fold related surfaces of IFN-� to form a 2:1 IFN-�R1/IFN-�complex (Fig. 3). Formation of this complex on the cell sur-face requires that the twofold of IFN-� be approximatelyparallel to the cell membrane. Although IFN-� oligomerizestwo IFN-�R1s, the receptors do not interact with one an-other and are separated by �100 Å, where they are predictedto enter the cell membrane.

The �-helical core of IFN-� bound to IFN-�R1 is essen-tially identical to the previously determined crystal structureof unbound IFN-� ; however, significant structural differ-ences are observed for the AB loop of IFN-� , which makesextensive contacts with IFN-�R1. These contacts result inwell-ordered AB loops that contain one turn of 310 helix. Incontrast, the AB loops in the crystal structure of unboundIFN-� exhibit highly variable conformations. Theconformational flexibility of this region is also supported byNMR studies. Although the mobility of the AB loop is influ-enced by IFN-�R1 binding, the positively charged C-terminal tail of IFN-� remains flexible and is not observedin the structure of the complex (22, 33, 34).

Although it is not observed in the structure, this region ofIFN-� is likely involved in receptor binding. It has beendemonstrated to affect receptor recognition and heparin sul-fate binding by BIAcore (35). Antibodies raised against thisregion of IFN-� block STAT1 alpha nuclear translocation, arequirement for biological activity (36). Mutation or deletionof the C-terminus affects receptor binding, dsRNA cleavage,

and antiviral activity (37, 38). Peptides corresponding to theC-terminus compete with IFN-� for receptor binding (39).

The extracellular fragment of IFN-�R1 folds into two do-mains to form a narrow rod-like molecule with overall di-mensions of 75 × 25 × 15 Å. The N-terminal (D1) and C-terminal (D2) domains are connected by a short linker thatadopts one turn of an �-helix. Unique to IFN-�R1, D1 andD2 are linked by a disulfide bond between Cys108 in the he-lical linker and Cys153 in D2. Approximately 1090 Å ofsurface area is buried in the D1–D2 interface that consistsmostly of hydrophobic residues. The interdomain angle be-tween D1 and D2 is approximately 120°. The IFN-� bindingsite is located in a crevice between D1 and D2.

The domains of IFN-�R1 are structurally similar tofibronectin type III domains (FBNIII). D1 adopts a FNBIII�-sandwich fold that is characterized by two �-sheets com-prised of �-strands A, B, and E and G, F, C, and C �. Al-though D1 of IFN-�R1 adopts a standard FBNIII topology,D2 contains an additional three residue �-strand (strand D)that is linked to �-strand E by a buried disulfide bond be-tween Cys181 and Cys186. �-Strand D forms part of the A,B, and E �-sheet, and with the exception of Cys181, its resi-dues display five solvent accessible glutamic and asparticacid residues on the receptor surface. The BC loop contrib-utes an additional five acidic residues that combine with �-strand D to form an acidic patch near the domain interface(Fig. 3). This patch has been hypothesized to form a tran-sient binding site for the basic C-terminal tail of IFN-�.

The structure of IFN-�R1 provided the first opportunityto compare the three-dimensional structures of class 1 andclass 2 cytokine receptors as defined by Bazan (31). Com-parison of IFN-�R1 with growth hormone receptor (GHR),the prototypical class 1 cytokine, revealed IFN-�R1 andGHR exhibit different domain angles of 120° and 90°, re-spectively. The functional consequence of the different do-main angles is that GHR displays six ligand binding loopscompared with five for IFN-�R1. Thus, the AB loop fromD1 participates in growth hormone binding in the GHR butis buried in the domain interface in IFN-�R1.

Residues of IFN-� in the binding interface are located ontwo discontinuous polypeptide segments. The first is com-prised of helix A, the AB loop, and helix B, while the secondcontains helix F. IFN-� is oriented with its N- and C-termininear the D1–D2 interface with the AB loop interacting withthe L2 loop on D1. A total of 960 Å2 of accessible surface isburied in each binding interface. The aromatic receptor resi-dues Tyr52 loop 2 (144 Å2), Trp210 loop 6 (95 Å2), andTrp85 loop 3 (75 Å2), contribute almost one third of the totalburied surface area. Tyr52 and Trp85 form a large protrudingsurface that makes extensive contacts with the AB loop,His111, and Glu112 of IFN-� . Of the eleven hydrogenbonds and salt bridges identified in the interface, five involvemainchain atoms of the AB loop. Near the end terminus ofthe molecule, IFN-� residues Val5 N termini, Ile114 helix F,and Ala118 helix F, pack against Trp210 loop 6 and Val209loop 6 of the receptor. In 2000, the crystal structure of thecomplex of human IFN-� with the soluble glycosylatedextracellular part of IFN-�R� revealed the presence of athird receptor molecule not directly associated with theIFN-� dimer (40).

© 2002 NRC Canada


Fig. 3. Molecular structure of IFN-� and its soluble high-affinityreceptor. The ligand dimer is colored as in Fig. 2; IFN-�R1 mol-ecules are in yellow and magenta.



The IL-10/IL-10R1 complexThe crystal structure of the IL-10/IL-10R1 complex shares

many similarities with the IFN-� /IFN-�R1 complex (41).Like IFN-� , IL-10 uses the same surfaces comprised of he-lix A, AB loop, and helix F to bind two IL-10R1s and forma 1:2 IL-10/IL-10R1 complex (Fig. 4). Despite differences inthe domain orientations of IL-10 and IFN-� , the separationbetween the C-terminal ends of the twofold related IL-10R1sis very similar to the IFN-� /IFN-�R1 complex (�110 Å vs.100 Å).

Comparison of bound and unbound IL-10 reveals a con-served �-helical core that does not change its structure uponreceptor binding. As observed for IFN-� , the AB loop of un-bound IL-10 is flexible but adopts a rigid conformation dueto several contacts with IL-10R1 in the receptor complex.Despite receptor binding the N-terminal ten and C-terminal two residues are not observed in the IL-10/IL-10R1complex and are assumed to be flexible.

IL-10R1 folds into two FBN-III domains to form an L-shaped molecule. The individual domains of IL-10R1 andIFN-�R1 are structurally similar. Comparison of D1 and D2domains results in root-mean-square deviations of 1.2 Å (81C� atoms) and 1.6 Å (71 C� atoms), respectively. Despitesimilar domain structures, IL-10R1 and IFN-�R1 exhibitmarkedly different interdomain angles of 90° and 120°, re-spectively. Despite the different domain angles, IL-10R1 andIFN-�R1 still use the same five loops (L2–L6) to bind theirrespective ligands; however, the positions of the L2–L3 D1loops change relative to the L5–L6 loops on D2. For IL-10R1,these loops are separated by �20 Å, while in IFN-�R1 thedistance is only 12 Å. These differences allow IFN-�R1 to

bind the short A and F helices in IFN-�, while IL-10R1 ismore suited to bind to the longer A and F helices of IL-10.

As a result, IL-10R1 contacts IL-10 at two distinct sur-faces labeled Ia and Ib. Site Ia occurs where the A–B loopcrosses helix F, while site Ib is located near the N-terminusof helix A and the C-terminus of helix F. Receptor bindingloops L2–L4 interact exclusively with site Ia, while loops L5and L6 in D2 interact with site Ib. As previously observedfor IFN-�R1, IL-10R1 residue Tyr43 located on loop 2 bur-ies the most surface area of any residue (�105 Å2) into ashallow cavity created by helix F and the AB loop.

Unique to the IL-10 system, IL-10 and sIL-10R1 form acomplex consisting of two IL-10 dimers and four IL-10R1molecules in solution. A 2:4 complex is also formed in theIL-10/IL-10R1 crystals by two adjacent 1:2 complexes thatseparated by unit cell translation of 46 Å and offset an addi-tional �21 Å in a direction approximately perpendicular tothe unit cell translation (Fig. 4). Contacts in the interface(site II) between the complexes are formed between IL-10R1D1 from one complex with IL-10 and IL-10R1 D2 of the ad-jacent complex. The location of the L2 loop in the site II aswell as the site I interface suggests that assembly of the 2:4is dependent on the initial formation of the site I interface(e.g., 1:2 complex). Sequence similarities between IL-10R1and IL-10R2 suggest both receptors may bind such that IL-10R2 may bind to the IL-10R1 binding site. This 2:4 com-plex provides a model for how IL-10R1 and IL-10R2 mayassociate to form a biologically active IL-10/IL-10R1/IL-10R2 complex.

Molecular models for IFN/IFN receptor complexesIFNAR1 and IFNAR2 are members of the class 2 cytokine

receptor family (31) permitting modeling of theirextracellular domains from the crystal structure of humanIFN-� receptor 1 (33, 40) or tissue factor (42). Several priormodels of IFNAR1 and IFNAR2 based upon IFN-� receptor1 and tissue factor have been presented (37–41).

Homology models were constructed for human IFNAR2and IFNAR1 based on the crystal structure of IFN-� receptor1 (33). Models were constructed for the two extracellulardomains of IFNAR2 (residues 1–202), extracellular domains1–2 of IFNAR1 (residues 1–202), and extracellular domains3–4 of IFNAR1 (residues 203–407). The alignment ofIFNAR1 domains and IFN-� receptor 1 was previously de-scribed by Cutrone and Langer (43). IFNAR2, IFNAR1 do-main 1–2, and IFNAR1 domain 3–4 models wereconstructed by the Segment Match Modeling Approach (44)and refined with the Encad program as implemented in theLook (Molecular Applications Group, formerly of Palo Alto,CA) program. The models were further refined by molecularmechanics minimization with the InsightII/Discover program(Accelrys, San Diego, CA).

The molecular model of IFNAR2 was docked manuallywith the InsightII program to the zinc dimer crystal structureof IFN-�2b (16) using the site and orientation previouslysuggested from mutational analysis (45–48). Priormutational analysis suggested key IFN-�2b–IFNAR2 interac-tions are D35–K50, R144–E79, R149–E79, S152–H78,R144–M48, and R22–Y45. Based on our docked orientationthe last interaction is likely indirect.

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Fig. 4. Molecular structure of IL-10/sIL-l0R1 (A) 1:2 and(B) 2:4 complexes. In plate (A) IL-10 monomers in cyan andgreen, while the sIL-10R1 molecules are in yellow and magenta.In plate (B) IL-10 monomers are in yellow, blue, cyan, and ma-genta, and sIL-10R1 molecules are in cyan and green.



The molecular models of the IFNAR1 domain 1–2 anddomain 3–4 were first docked to the IFN-�2b crystal struc-ture independently and were later annealed to form a peptidebond between domains 2 and 3 with residues mapped bymutagenesis as forming the IFNAR1 binding site on thesame side of the model as suggested from prior modeling(43). Mapping of bovine IFNAR1 binding residues in thatwork demonstrated the importance of IFNAR1 domains 2and 3 (49). Domain 2 residues mapped by loss of bindingmutations included W132, F139, Y141, and Y160, while asingle domain 1 residue Y71 and a single domain 3 residueW253 appeared important as well. Of residues previouslynoted to form the IFNAR1/IFN-� recognition site, Y71,W132, and Y160 of the first two domains appear to form anavailable surface for docking, with F139 and Y141 proppingup these positions. Prior IFN-� chimers and mutations sug-gest the IFNAR1 recognizes IFN-� AB loop residues 16–28as well as K83 and Y90 (50). Given the expected similar ori-entation of IFNAR1 and IFNAR2 transmembrane segments,we oriented the IFNAR1 domain 1–2 model along the ABloop and the domain 3–4 model against IFN-� K83 andY90. Specific interactions could be formed in docking be-tween IFN-�2b–IFNAR1 S23 and S25–Y71, R22–W132,K83–Y160, and K83–W253. The resulting annealed IFNAR1model contain an approximately perpendicular angle betweendomains 2 and 3. The resulting IFN-�2b zinc dimerIFNAR1/IFNAR2 complex model was subject to further re-finement by molecular mechanics minimization withInsightII/Discover.

The docked orientation of the IFN-�2b/IFNAR1/IFNAR2complex is presented in Fig. 5. IFNAR1 and IFNAR2 do nothave a significant contact region excepting the N-terminus ofIFNAR2. The association of IFNAR1 and IFNAR2 does not

require IFN-�2b dimer formation in this orientation. Chime-ric constructs using the extracellular domain of the EPO re-ceptor and transmembrane and intracellular regions ofIFNAR1 demonstrate receptor dimerization is required forinduction of effector genes (51). A similar construct with theextracellular domain of IFN-�R1 and transmembrane andintracellular regions of IFNAR2 shows receptor dimerizationis sufficient to induce biological activity (52). Thus, liganddimerization, perhaps mediated by zinc, may permit a com-plex to form with two molecules of IFNAR1 and IFNAR2.These receptors could recognize identical sites on the dimerin a manner analogous to IFN-�R1 (33).

IFNAR2 dimerization may be a requirement for antiviralactivity. Both IFNAR2 and IFNAR1 are subject to alterna-tive splicing, producing soluble-receptor isoforms. Differen-tial regulation of IFNAR2 isoform expression has beenrecently suggested as a means of modulating type I IFN re-sponses (53). While the intracellular domain of IFNAR2 isrequired for signal transduction, interaction of its solubleisoform, containing only the extracellular domain, with itsmembrane anchored isoform is sufficient to confer antiviralactivity even in the absence of type I IFN (54). The nature ofthis interaction is unclear as direct contact between IFNAR2extracellular domains is not seen in our proposed model (seeFig. 6).

Although, it is perceived that IFN-�2 exists as a monomerat physiologically relevant concentrations, local concentra-tion at the receptor or receptor mediated association may ob-ligate dimer formation. NMR and dynamic light-scatteringstudies have shown IFN-�2 exists in a oligomeric rather thanmonomeric state at neutral pH (19, 55). Since IFN-� and

© 2002 NRC Canada


Fig. 5. The IFN-� 2b/IFNAR1/IFNAR2 complex. IFN-� 2b isshown in cyan, IFNAR1 in green, and IFNAR2 in red.

Fig. 6. The IFN-� 2b/IFNAR1/IFNAR2 dimer model. Coloring asin Fig. 5; zinc is indicated by a yellow sphere.



IL-10 activate their respective receptors as dimers, we usedthe dimer of IFN-�2b to generate symmetrical counterpartsfor IFNAR1 and IFNAR2 on the IFN-�2b dimer. This orien-tation is shown in Fig. 6. From this model, it is evidentIFN-�2b dimers could recognize two copies of IFNAR1 andIFNAR2.

IFN-� can form a dimer with zinc as well, although themonomer orientation differs from IFNA2 (56). TheIFNAR1/IFNAR2 complex also appears to recognize IFN-�at a different location on the ligand framework (56).

Conclusion

The proposed IFN-�2b/IFNAR1/IFNAR2 model of a dimerligand binding to a pair of receptors on each of the chains ofthe ligand is consistent with the model proposed for thebinding of cytokines to class 2 receptors.

Acknowledgments

We thank Gideon Schrieber for providing Proofs of theIFN-�2b/IFNAR2 complex paper prior to publication. Wethank Patricia Weber and Johanthan Greene for help in thepreparation of this manuscript.

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type i interferon structures: possible scaffolds for the interferon-alpha receptor complex

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