223 IL22R, IL10R2 and IL22BP binding sites are topologically juxtaposed on adjacent and overlapping...

doi:10.1016/j.jmb.2008.07.046 J. Mol. Biol. (2008) 382, 1168–1183

Available online at www.sciencedirect.com

IL-22R, IL-10R2, and IL-22BP Binding Sites AreTopologically Juxtaposed on Adjacent and OverlappingSurfaces of IL-22

Paul W. Wu1†, Jing Li2†, Sreekumar R. Kodangattil2,Deborah P. Luxenberg1, Frann Bennett1, Margot Martino1,Mary Collins1, Kyriaki Dunussi-Joannopoulos1, Davinder S. Gill2,Neil M. Wolfman1 and Lynette A. Fouser1⁎
1Wyeth Research—Inflammation,Cambridge, MA 02140, USA2Wyeth Research—BiologicalTechnologies, Cambridge,MA 02140, USA
Received 10 April 2008;received in revised form16 July 2008;accepted 17 July 2008Available online25 July 2008

*Corresponding author. E-mail [email protected].† P.W.W. and J.L. contributed equPresent addresses: J. Li, Biologics, O

3C-252,Novartis Institute for BiomedMassachusetts Avenue, Cambridge,S. R. Kodangattil, PfizerGlobal ReseaEastern Point Road, Groton, CT 0634Abbreviations used: IL, interleuki

ECD, extracellular domain; IL-22BP,H/F, hexahistidine/FLAG® octapephorseradish peroxidase; PDB, Protei

0022-2836/$ - see front matter © 2008 E

Interleukin (IL) 22 is a type II cytokine that is produced by immune cellsand acts on nonimmune cells to regulate local tissue inflammation. As aproduct of the recently identified T helper 17 lineage of CD4+ effectorlymphocytes, IL-22 plays a critical role in mucosal immunity as well as indysregulated inflammation observed in autoimmune diseases. We usedcomprehensive mutagenesis combined with mammalian cell expression,ELISA cell-based, and structural methods to evaluate how IL-22 interactswith its cell surface receptor, IL-22R/IL-10R2, and with secreted IL-22binding protein. This study identifies those amino acid side chains of IL-22that are individually important for optimal binding to IL-22R, considerablyexpands the definition of IL-22 surface required for binding to IL-10R2, anddemonstrates how IL-22 binding protein prevents IL-22R from binding toIL-22. The IL-22R and IL-10R2 binding sites are juxtaposed on adjacentIL-22 surfaces contributed mostly by helices A, D, and F and loop AB.Our results also provide a model for how IL-19, IL-20, IL-24, and IL-26which are other IL-10-like cytokines, interact with their respective cellsurface receptors.

© 2008 Elsevier Ltd. All rights reserved.

Edited by I. Wilson
Keywords: IL-22; structure/function; IL-22R; IL-22BP; IL-10R2
Introduction

Interleukin (IL) 22 is a member of the IL-10-likesubgroup of type II cytokines, the latter alsoincluding interferons (IFNs).1 It is produced byactivated T helper 17 CD4+ lymphocytes as well as

ess:

ally to this work.ncologyDepartment,ical Research, Inc., 250MA 02139, USA;rch andDevelopment,0, USA.n; IFN, interferon;IL-22 binding protein;tide; HRP,n Data Bank.

lsevier Ltd. All rights reserve

dendritic cells, with its expression highly depen-dent on IL-23.2,3 IL-22 is known to regulate localtissue inflammation, in spite of its action on onlynonimmune cells.4–8 Recent clinical and/or preclin-ical studies suggest that IL-23-dependent produc-tion of IL-22 is critical to mucosal immunity in thelung and gut9,10 and to progression of psoriasis, ahuman autoimmune disease of the skin.3,11–15 IL-22induces hyperproliferation of skin keratinocytesand resultant thickening of the epidermis, whichare both characteristics of psoriatic lesions.16 Inaddition, IL-22 induces gene expression fromkeratinocytes that appears to be critical for recruit-ment of immune cells and maintenance of psoriatictissue inflammation.15–17

At low concentrations, IL-22 exists as a monomerin solution, sharing a six α-helix structural andfunctional monomeric unit with the intercalatedIL-10 dimer, as well as other IL-10-like cytokinesand IFN-γ.18–22 The cell surface receptor for IL-22consists of the IL-22R and IL-10R2 subunits, both

d.

1169IL-22's IL-22R, IL-10R2, and IL-22BP Binding Sites

present on epithelial cells and some fibroblastswithin various tissues.6,23–26 Although IL-22R andIL-10R2 individually contribute to other type IIcytokine receptors, these two subunits together arespecific for only IL-22. Thus, IL-22 induces distincteffects on a cell via its unique receptor. IL-22 bindsfirst to the extracellular domain (ECD) of IL-22R.19,27

Due to a proposed IL-22R-induced conformationalchange in IL-22, IL-10R2 is able to bind to an IL-22/IL-22R surface.27,28 The resultant IL-22/IL-22R/IL-10R2 complex, either as a heterotrimer or as amultimer thereof, is then able to transmit a signalinto a cell via the JAK/STAT and MAPK signalingpathways.29–31

IL-22 also binds to the IL-22 binding protein(IL-22BP), a secreted ‘receptor’ that is specific forIL-22 and has 33% primary sequence identity tothe ECD of IL-22R.32 A cell surface form of IL-22BPhas not been identified. In vitro, IL-22BP acts as adecoy receptor and blocks IL-22 signaling into acell.32,33There is limited understanding of how IL-22

interacts with its cell surface receptor and bindingprotein. A focused mutagenesis study demonstratedthat six amino acids in IL-22 helices A and D, inclu-ding a glycosylated asparagine, are required foroptimal binding to IL-10R2.28 The relevance of helixA and helix D peptides to IL-10R2 binding wassubsequently substantiated.34 Residues within heli-ces A and F and loop AB of IL-22 have been pro-posed to be important for binding to a receptorbased on superimposition of IL-22 structure tocytokine within IL-10/IL-10R1ECD or IFN-γ/IFN-γR1ECD co-crystal structures, as well as inferencesfrom an IL-22/IL-22RECD model.19,20 However,IL-22 side chains that are important for binding toIL-22R and IL-22BP have not been experimentallydetermined.To better define the surfaces of IL-22 that are re-

cognized by its cellular and soluble receptors, wegenerated and studied a comprehensive collectionof IL-22 mutant proteins corresponding to a subs-titution at each position in mature IL-22. The func-tional evaluation of these 146 point substitutionsdetermined that 29 side chains are individuallycritical for optimal binding of IL-22 to IL-22BP,IL-22R, and/or IL-10R2. Our findings define sidechains important for binding to IL-22R and IL-22BP,expand our understanding of how IL-22 interactswith IL-10R2, and demonstrate how IL-22BP inter-feres with the interaction between IL-22 and its cellsurface receptor. Adjacent surfaces on IL-22, con-tributed mostly by helices A, D, and F and loop AB,are required for the temporal development of theIL-22/IL-22R and IL-22/IL-22R/IL-10R2 com-plexes. IL-22 is the first IL-10-like cytokine to becomprehensively mutagenized for the study offunction in relation to structure. We propose thatour elucidated receptor binding sites for IL-22 canbe transposed to the structurally homologous re-gions of IL-19, IL-20, IL-24, and IL-26 and are anapproximation of these cytokines' high-affinity andlow-affinity receptor binding sites.

Results

A set of 146 distinct point substitutions in IL-22is efficiently evaluated for an effect on IL-22receptor and antibody binding

A panel of IL-22 mutants was generated by amethod employing three polymerase chain reactions(PCRs) for each point mutation. The product of thethird PCR was a mutated human IL-22 linear DNAexpression cassette. This 1816-bp linear DNA en-coded a mammalian promoter, translation start, andsecretory leader, with the latter fused in-frame to anN-terminal hexahistidine/FLAG® octapeptide(H/F)-tagged IL-22, followed by a polyadenylationsequence. Using this PCR-based method, the sevenalanines of the mature IL-22 open reading framewere individually mutated, either to glycine or to adifferent alanine codon. The remaining amino acidsof IL-22 were individually changed to alanines.These IL-22 linear DNA expression cassettes wereused for transient expression in mammalian cellsand IL-22 in the conditioned media quantitated bysandwich ELISA, exploiting both entities of the H/Ftag at the N-terminus of the cytokine.To determine which IL-22 amino acids are im-

portant for binding to IL-22 cell surface and solublereceptors, we evaluated our panel of IL-22 pointsubstitutions in five distinct ELISAs that useIL-22BP-Fc and IL-22R-Fc homodimers, IL-22R-Fc/IL-10R2-Fc heterodimers, or IL22-02 and IL22-04antibodies to capture the IL-22 cytokine in the con-ditioned media.27 The two antibody assays, whichdetect distinct IL-22 epitopes (data not shown), wereused in concert with the three IL-22 receptor assaysto identify those IL-22 mutations that affect theoverall stability of IL-22 secondary and/or tertiarystructures and only indirectly affect receptor orantibody binding. Approximately 800 independentdata points that described the binding characteris-tics of single amino acid substitutions spanning themature primary sequence of IL-22 were collected induplicate.The binding of silent alanine substitutions derived

from 13 separate transfections served as the controlIL-22 cytokine. The signal threshold for the bindingof control IL-22 was set at 1.77 standard deviations(SD) below the mean signal in a given assay and is aone-sided 95% confidence interval for individualobservations. IL-22 mutants that gave a bindingsignal below the threshold were defined as beingsuboptimal for binding to a given receptor orantibody.Sixty-five of the 146 (45%) alanine substitutions

bound comparably to control IL-22 in the five IL-22receptor and antibody assays. Ranging from entirelysurface-exposed to buried, the individual integrityof the corresponding IL-22 residues was notessential for optimal binding to IL-22R, IL-10R2, orIL-22BP.Thirty of the 146 (20%) substitutions had statis-

tically significant suboptimal binding, compared to

1170 IL-22's IL-22R, IL-10R2, and IL-22BP Binding Sites

silent mutants, in four (10 mutants) or five (20mutants) assays (Table 1 and Fig. 1). The strongestinhibition in all five assays was observed with thealanine substitution of I75, L100, C132, E166, andC178. We propose that the 10 additional substi-tutions classified as statistically significant in onlyfour assays (Table 1) also had a subtle impact onthe IL-22R binding assay (Fig. 1, 'near'). Consider-

Table 1. The effect of IL-22 point mutations on the general st

a In 1M4R IL-22 structure at a probe radius of 1.4 Å.b Fold change in binding is relative to purified human IL-22, with e

tration as purified IL-22 in a given assay.c The 13 mutations in gray are silent and were used to establish the

interval) for each assay. A mutation would then need to bind below thfor binding to a given receptor or antibody.

d Mutations in bold had suboptimal binding to all receptor Fc ande The two mutations in enlarged and bold text were purified for fuf Mutations not in bold against a white background had suboptimal

the threshold (see also 'near' column in Fig. 2a).

ing that relatively few of the 30 corresponding IL-22side chains are exposed to the surface (see solventaccessibility values in Table 1), we concluded thatthese IL-22 side chains do not contribute specifi-cally to the receptor binding sites. Rather, wepropose that these 30 amino acids are importantfor maintaining IL-22 secondary and/or tertiarystructures.

ability of IL-22 structure

ach conditioned medium tested in duplicate at the same concen-

binding threshold (mean−1.77 SD for a one-sided 95% confidenceis threshold in a given assay in order to be defined as suboptimal

IL-22 antibodies.rther analysis.binding in four assays with binding to IL-22R, the fifth assay, near

Fig. 1. Systematic mutagenesisof human IL-22 reveals specificamino acids that are critical for theglobal stability of cytokine struc-ture. One hundred forty-six pointsubstitutions and 13 silent muta-tions in IL-22 were evaluated at asingle concentration in each of fivebinding ELISAs, all normalized tothe binding of purified IL-22. Thecomplete set of binding data for ascreen of IL-22-conditioned mediais shown in Supplementary Table 1.A subset of these data (Table 1) thatis focused on the 30 mutationscollectively defined as affectingglobal IL-22 structure is graphicallydisplayed here, along with data forthe silent substitutions. The solidsymbols correspond to those pointsubstitutions that were determinedto be suboptimal for binding(below) relative to the silent sub-stitutions in four or five assays. The

horizontal bars indicate the threshold for suboptimal binding, which is 1.77 SD below the mean value for the silentsubstitutions (open symbols: ‘silent’). The range in values for the silent mutants is due to experimental error in thequantitation of the tagged IL-22 protein within the conditioned media samples. Broader value ranges for the silentsubstitutions also correlate with the lowest-affinity interactions (i.e., IL-22R, IL22-02, and IL-22R/IL-10R2). While notsuboptimal for binding, 10 of the point mutants bound at, or immediately above, the IL-22R threshold (semisolidtriangles: ‘near’). We hypothesize that the calculated threshold for the IL-22R silent mutations was set too low, as a resultof experimental error in protein quantitation and the relatively low affinity of the IL-22/IL-22R interaction, and that thissubset of substitutions also has some suboptimal effect on binding to IL-22R.


The integrity of 29 side chains in IL-22 isrequired for optimal binding to IL-22BP, IL-22R,and/or IL-10R2

Twelve of the 146 (8%) IL-22 residues weredetermined to be critical for binding to IL-22R,since alanine point mutants of these did not bindas well as control silent mutants in the IL-22Rbinding assay (red and blue residues or solidsymbols in Table 2 and Fig. 2). Alanine substitutionof I161, V72, G165, D71, and L169 had the mostdeleterious effect on the IL-22R-Fc binding assay.Three of these 12 substitutions (D67A, R73A, andK162A) also did not bind as well as control IL-22mutants to IL-22BP (blue residues and solidsymbols in Table 2 and Fig. 2), indicating that thecorresponding IL-22 side chains are critical foroptimal binding to IL-22BP. Collectively, these datasuggest that the IL-22R and IL-22BP binding siteson IL-22 are overlapping.We observed that 17 of the 146 (12%) residues in

IL-22 are critical for optimal binding to IL-10R2.Twenty-nine amino acid substitutions did not bind aswell as control IL-22 in the IL-22R-Fc/IL-10R2-Fcbinding assay. Logsdon et al. and Li et al. havedemonstrated previously that IL-10R2 binds to apreformed IL-22/IL-22R complex.19,27 As 12 of these29 mutants were also less effective in the IL-22R-Fcbinding assay, we inferred that the remaining 17mutants correspond to IL-22 side chains that are

specifically critical for binding to IL-10R2 (yellow andgreen residues or solid symbols in Table 2 and Fig. 2).Alanine substitution of T56, Y51, R55, N54, F121, andE117 had the most deleterious and specific effect onthe IL-22R-Fc/IL-10R2 binding assay. The alaninesubstitution of V83 bound less well in both IL-22R-Fc/IL-10R2-Fc and IL-22BP-Fc assays (green residueor solid symbol in Table 2 and Fig. 2), indicating thatthe corresponding IL-22 side chain may be importantfor both IL-10R2 and IL-22BP binding.None of the 146 point substitutions in IL-22 had a

uniquely deleterious effect on the IL-22BP bindingassay. Rather, the four IL-22 side chains that werecritical for optimal binding to IL-22BP were alsoimportant for binding to IL-22R or IL-10R2 (blue andgreen residues/solid symbols in Table 2 and Fig. 2,respectively).To validate the initial screen, which used IL-22

point mutants in conditioned media to study bind-ing characteristics, we chose 13 point substitutionsfor further evaluation subsequent to purification.Nine of these blocked IL-22 binding to IL-22BP,IL-22R, or IL-22R/IL-10R2 (Table 2), two affectedglobal stability of structure (Table 1), and two hadno effect on binding (Supplementary Table 1) in theinitial screen. The solvent accessibility of the IL-22residues corresponding to these substitutions rangesfrom 3% to 71%. The binding data collected withpurified IL-22 point mutants (Fig. 3) confirmed thesuboptimal effects on binding observed in the initial

Table 2. The effect of IL-22 point mutations on binding to receptor Fc

a In 1M4R IL-22 structure at a probe radius of 1.4 Å.b Fold change in binding is relative to purified human IL-22, with each conditioned medium tested in duplicate at the same concen-

tration as purified IL-22 in a given assay.c The 13 mutations in gray are silent and were used to establish the binding threshold (mean−1.77 SD for a one-sided 95% confidence

interval) for each assay. A mutation would then need to bind below this threshold in a given assay in order to be defined as suboptimalfor binding to a given receptor.

d Mutations in red were suboptimal for binding to both IL-22R and IL-22R/IL-10R2.e The nine mutations in enlarged and bold text were purified for further analysis.f Mutations in blue were suboptimal for binding to both IL-22R and IL-22BP.g Mutations in yellow were suboptimal for binding to only IL-10R2.h Mutation in green was suboptimal for binding to IL-10R2 and IL-22BP.


high-throughput screen. IL-22 mutants that con-tained alanine substitution of D67, V72, R73, I161,K162, or L169 were approximately 50-fold less effec-tive than control cytokine for binding to IL-22R-Fc(Fig. 3a, red and blue symbols and lines). Thosemutants that blocked binding to IL-22R were alsoless effective to varying degrees in the IL-22R-Fc/IL-10R2-Fc binding assay (Fig. 3b, red and blue). As

expected, three of these purified IL-22 substitutions(D67A, R73A, and K162A) were also deleterious forbinding to IL-22BP-Fc (Fig. 3c, blue symbols andlines). Mutants that were specifically defective forbinding to IL-10R2 in the initial screen were alsosuboptimal for binding to IL-22R-Fc/IL-10R2-Fcwhen purified and evaluated at different concen-trations (Fig. 3b, yellow) and bound optimally to

Fig. 2. Systematic mutagenesisof human IL-22 reveals specificamino acids that are critical foroptimal binding to IL-22R, IL-10R2, and IL-22BP. (a) One hundredforty-six point substitutions and 13silent mutations in IL-22 were eval-uated at a single concentration ineach of five binding ELISAs, allnormalized to the binding of pur-ified IL-22. The complete set ofbinding data for a screen of IL-22-conditioned media is shown inSupplementary Table 1. A subsetof these data (Table 2) that isfocused on the 29 mutants (solidsymbols), collectively defined assuboptimal for binding to IL-22R(red and blue), IL-10R2 (yellow andgreen), or IL-22BP (blue and green),is graphically displayed here alongwith data for the silent substitu-tions. The horizontal bars indicatethe threshold for suboptimal bind-ing, which is 1.77 SD below themean value for the silent substitu-tions (open symbols: ‘silent’). Therange in values for the silentmutants is due to probable error inthe quantitation of the tagged IL-22protein within the conditionedmedia samples. Broader valueranges for the silent substitutionsalso correlate with the lowest-affi-nity interactions (i.e., IL-22R and IL-22R/IL-10R2). (b) The mature pri-mary sequence of IL-22 is shownwith numbering initiated from theN-terminus of the secretory leader.The dashed lines indicate intramo-lecular disulfide bonds, while thestick models indicate putative gly-cosylation of asparagines at posi-tions 54, 68, and 97. (c) Ribbonrendering of the IL-22 tertiary back-bone is shown, with helices A–Fannotated. Amino acids defined ascritical for binding to IL-22R (redand blue), IL-10R2 (yellow andgreen), and IL-22BP (blue andgreen) are highlighted in both (b)and (c).


IL-22R-Fc or IL-22BP-Fc (Fig. 3a and c, yellow sym-bols and lines). We conclude that the data collectedwith the initial screen were predictive of observa-tions that were subsequently obtained with purifiedmutants.

In summary, the individual integrity of 12 and 17IL-22 side chains was required for optimal bindingto IL-22R and IL-10R2, respectively. The integrity offour IL-22 amino acids was required for optimalbinding to IL-22BP.

Fig. 3. IL-22 point substitutions in helices A, D, and Fand loop AB bind suboptimally to IL-22BP, IL-22R, and/orIL-22R/IL-10R2. Purified H/F–IL-22 mutants were eval-uated for binding to plates coated indirectly with IL-22R-Fc (a), IL-22R-Fc/IL-10R2-Fc (b), and IL-22BP-Fc (c), usingHisProbe HRP to detect the His-tag at the N-terminus ofthe cytokine. Nine IL-22 substitutions that exhibitedsuboptimal binding to IL-22BP (blue), IL-22R (red andblue), or IL-10R2 (yellow) in the systematic high-through-put assays were evaluated as were two that had anadverse effect on all five binding assays (solid black) andtwo that had optimal binding characteristics in all fiveassays (open black). The binding of purified IL-22 with nomutations is shown as a dashed line. Data are representa-tive of at least two experiments.

Fig. 4. IL-22 point substitutions in helices A, D, and Fand loop AB suboptimally induce the proliferation of cells.IL-22-dependent proliferation of BaF3 cells expressingboth receptor subunits was evaluated after 72 h by [3H]thymidine incorporation. Nine IL-22 substitutions thatexhibited relatively poor binding to IL-22R (red and blue)or IL-22R/IL-10R2 (yellow) in the ELISA-based receptorbinding assays were evaluated. In addition, two IL-22mutants that had an adverse effect on all five assays (solidblack) as well as two IL-22 mutants that had normalbinding characteristics in all five assays (open black) wereevaluated. The binding of IL-22 with no mutations isshown as a dashed line. Data are representative of at leasttwo experiments.


IL-22 side chains required for optimal binding toIL-22R or IL-10R2 are also required for signalinginto a cell

The purified IL-22 substitutions were also eva-luated for the ability to induce the proliferation of an

IL-22-dependent BaF3 cell line that overexpressedboth human IL-22R and IL-10R2 (Fig. 4). The 11mutants that did not bind as well as control IL-22 toIL-22R or IL-10R2 in the ELISA binding assays (Fig.3a and b) were, in general, similarly suboptimal forinducing IL-22-dependent cell proliferation (Fig. 4).One exception was the R73A substitution thatbound poorly to IL-22R-Fc/IL-10R2-Fc (Fig. 3b,R73A in blue). This same IL-22 mutant effected asignal into a cell when a 40-fold-higher concentra-tion was added relative to control IL-22 (Fig. 4, R73Ain blue). The two other exceptions were the alaninesubstitutions of I161 and L169. In the IL-22R-Fc/IL-10R2-Fc binding assay, the I161A mutant IL-22 wasa stronger binder than the V72A and L169Amutants(Fig. 3b). In contrast, in the IL-22-dependent cell-based assay, the V72A, I161A, and L169A substitu-tions were similarly less effective at lower concen-trations, with the L169A substitution inducing moreproliferation at higher concentrations (Fig. 4; datanot depicted). Overall, however, the above studies ina cell-based assay indicate that how effectively anIL-22 mutant interacts with IL-22R and IL-10R2 byELISA is quite predictive of its effectiveness insignaling into a cell.

Side chains mostly in IL-22 helices A, D, and Fand loop AB are required for optimal recognitionby IL-22BP, IL-22R, and/or IL-10R2 and definethe cell surface receptor binding sites

To explore the structural implications of our obser-vations, the IL-22 residues that were important foroptimal cell signaling and/or receptor binding wereconsidered in the context of existing IL-22 crystal


structure.20,35 As shown in the primary sequenceand ribbon rendering of the structure (Fig. 2b and c),the proposed IL-22R binding site is defined by

critical IL-22 side chains within helices A and F, andloop AB (red and blue substitutions). The IL-10R2binding site is adjacent to and defined by IL-22 sidechains in helices A and D, with contributions fromloops AB, BC, and CD, and helices C and F (yellowand green). The binding site for IL-22BP requires theintegrity of four IL-22 side chains (blue and green),three of which are in loop AB and helix F (blue) andtransect the proposed IL-22R binding site. Sequen-tial 90° rotations of IL-22, to the right around thevertical axis of the Corey-Pauling-Kendrew (CPK)models in Fig. 5, emphasize the restriction of the IL-22 cell surface and soluble receptor binding sites tocertain surface regions of the IL-22 structure.

Discussion

We report the systematic mutagenesis of the 146amino acids in IL-22 and the impact of each substi-tution on the binding of IL-22 to its cell surface re-ceptor and secreted binding protein, demonstratingthat the effects on receptor binding are predictive ofimpact on signal transduction. We define 12 IL-22side chains needed for optimal binding to IL-22R, 17side chains, including 5 side chains previouslyreported,28 required for binding to IL-10R2, and 4side chains needed for optimal binding to IL-22BP.We demonstrate that the high-affinity IL-22R bind-ing site and the low-affinity IL-10R2 binding site arecontiguous to each other and localized, incorporat-ing IL-22 side chains from helices A, D, and F andloop AB with contributions from helix C and loopsBC and CD. We conclude that the IL-22BP bindingsite overlaps with the IL-22R binding site, demon-strating at the structural level how this proposeddecoy receptor blocks a functional interaction bet-ween IL-22 and IL-22R.This study of IL-22 is the first to explore by com-

prehensive mutagenesis a structure/function rela-tionship for an IL-10-like cytokine. The members ofthis group (i.e., IL-10, IL-19, IL-20, IL-22, IL-24, andIL-26) are proposed to have a conserved six α-helixstructural and functional unit that is also sharedwith the IFNs.1,36 The receptors for these cytokinesand the IFNs belong to cytokine receptor family 2.Structural elucidation and modeling studies sug-gest that these receptors also share a conservedstructure.1,36

Glycosylated human IL-22 that is expressed andpurified from insect cells is proposed to function as amonomer.19 A recent report indicates that Escherichiacoli-derived IL-22 dimerizes at high concentrations

Fig. 5. The IL-22 amino acids that are critical forbinding to IL-22R, IL-10R2, and IL-22BP contribute toadjacent and overlapping binding sites on the surface ofIL-22. (a–d) Sequential 90° rotations to the right aroundthe vertical axis of a CPK rendering of IL-22. Coloredshading indicates those amino acid side chains that weredefined as critical for binding to IL-22R (red and blue), IL-10R2 (yellow and green), and IL-22BP (blue and green).


via a proposed interaction between its DE loops andis able to associate with the IL-22R receptorsubunit.37 The resultant low-resolution quaternarystructure is similar to that observedwith IL-10 dimerand its high-affinity receptor, IL-10R1.38 Singlesubstitutions of amino acids in the DE loop of IL-22, derived from mammalian cells, did not reducebinding to either IL-22R or IL-22R/IL-10R2 in ourassays. This indicates that none of the DE loopresidues is singularly critical for receptor binding.The ribbon rendering in Fig. 2c shows, however, thatdimerization via the DE loop would be quiteremoved from the receptor binding sites and, there-fore, compatible with our model for how IL-22interacts with its receptors.

Fig. 6. A structure-based alignment of IL-22 and IL-10 seque22 and IL-10 structure/function and is a simplification of Suppsequence was derived from the superimposition of IL-10, IL-19is IL-22, with amino acids determined to be critical for bindingIL-22BP (blue and green) highlighted. The second line of sequeto certain amino acids that have been previously demonstrated10R2 (green).39 The cylinders show the positions of the IL-22dashed bars below indicating the regions that contribute to IL-relation to IL-10's IL-10R1 (blue)38,39 and IL-10R2 (green)39 bidashed bars agree with the corresponding annotation of IL-22

IL-22R binding is dependent on the integrity ofIL-22 side chains in helices A and F and loop AB

IL-22 binds first to IL-22R, its high-affinity re-ceptor subunit.19,27 The 12 IL-22 amino acids iden-tified in this study to be important for optimalbinding to IL-22R are located in helix A (F57 andL59), loop AB (D67, T70, D71, V72, and R73), andhelix F (G159, I161, K162, G165, and L169) (Fig. 2band c). Eight of these have 12–86% solvent accessi-bility (see Table 2), suggesting that each maycontribute atoms to the IL-22R receptor interface.The remaining four amino acids (i.e., L59, G159,I161, and K162) are almost or completely buried,indicating that these contribute indirectly by facili-

nce and receptor binding sites. This figure is focused on IL-lementary Fig. 1. The alignment of IL-22 and IL-10 primary, and IL-22 monomeric structures. The first line of sequenceto IL-22R (red and blue), IL-10R2 (yellow and green), andnce is IL-10, with the short bars underneath correspondingto be important for binding to IL-10R1 (blue)38,39 and IL-and IL-10 helices in relation to the above sequence, with22's IL-22R (red) and IL-10R2 (purple) binding sites and innding sites. The numbering and coloring of residues andand IL-10 structures in Figs. 7b and 8b.


tating a local surface structure. The proposed IL-22Rbinding site is illustrated in CPK (Fig. 5a–d, red andblue residues) and solvent accessibility (Fig. 7a, redresidues) renderings.With superimposition of their IL-22 crystal

structure to the cytokine within IL-10/IL-10R1ECDand IFN-γ/IFN-γR1ECD co-crystal structures,Nagem et al. proposed that T70 and D71, as wellas four other side chains of IL-22, are importantfor the recognition of IL-22's corresponding high-affinity receptor subunit.20 Logsdon et al.,19 usinga model of IL-22/IL-22R based on their IL-10/IL-10R1ECD co-crystal structure,38 subsequently pro-posed that D71, R73, and G165, as well as sevenother side chains of IL-22, may contribute to IL-22R binding. Our study proves experimentallythat four of these previously proposed side chains(i.e., T70, D71, R73, and G165), as well as the eightadditional side chains that we have identified, arecritical for the generation of the optimal IL-22Rbinding interface. We note that five amino acidsproposed by Logsdon et al. are surface neighbors(noncolored labeled residues in Fig. 7a) to theresidues that we identified as contributory (Fig.7a, red residues).19 While not singularly as criticalto binding as those we have identified, it isfeasible that K61, S64, N68, E166, and D168 mayalso contribute to the IL-22R binding interface.The high-affinity receptor binding interface for IL-

10 was elucidated from the IL-10/IL-10R1ECD co-crystal structure.38,39 The IL-10R1 binding site en-compasses much of IL-10's helix A, loop AB, andhelix F (see IL-10R1-a and IL-10R1-b dashed bluebars and residues in the structurally derived pri-mary sequence alignment of Fig. 6, and the super-imposed stereo rendering in Fig. 7b). Nine of the IL-22R critical amino acids align with the IL-10R1-aregion. The three remaining residues (i.e., F57,G159, and I161) are immediately to the N-terminalside of the IL-10R1-a sites in helices A and F (Fig. 6).We propose that the IL-22R binding site is slightlyexpanded in helix A and translated upstream inhelix F of IL-22 (red dashed bars in Fig. 6) relative tothe IL-10R1-a binding site in these same helices ofIL-10.None of the IL-22 amino acids that align with

the IL-10R1-b regions of IL-10 (Figs. 6 and 7b)was determined to be critical for IL-22's interac-tion with IL-22R. This dissimilarity may be due tothe use of different methods, as described above,to explore the structure/function of IL-10 and IL-22. It would be interesting to know whether IL-10substitutions in the IL-10R1-b region of IL-10would be identified by our mutagenic methods.Alternatively, our results may reflect a morecompact IL-22R binding site than the correspond-ing IL-10R1 binding site on IL-10, as shown bycomparing the IL-22 (red) and IL-10 (blue) sidechains in the superimposed stereo rendering ofFig. 7b. In consideration of the distinct methodsused to study the interaction between IL-10 or IL-22 and their respective high-affinity receptors, weconclude that gross conservation of function as

dictated by structure exists between these twocytokines.

IL-22 surface structure that is required for IL-22Rbinding is also needed for IL-22BP

IL-22BP specifically blocks an interaction betweenIL-22 and IL-22R, suggesting that IL-22BP has atleast a partially overlapping binding site with IL-22R.27 We demonstrate that the integrity of four IL-22 side chains in loops AB and BC, and helix F isneeded for optimal binding to IL-22BP and IL-22R,and we propose that D67, R73, and K162 contributedirectly to these binding sites (Fig. 7a and c). Theimportance of V83 is probably indirect, since it hasonly 3% solvent accessibility. These four aminoacids of IL-22 are not sufficient for binding to IL-22BP; IL-22BP is specific for IL-22, yet these samefour amino acids are conserved between IL-22 andIL-24. In consideration of IL-22BP's high affinity forIL-22 (data not shown), we propose that other IL-22side chains in the vicinity of D67, R73, and K162 (seeFig. 7c) also contribute to the IL-22BP binding sitebut are not as singularly critical and, thus, were notdetected with our experimental methods. Theexistence of IL-22BP, its specificity and high affinityfor IL-22, and the fact that it interferes with IL-22binding to IL-22R, by a mechanism proposed above,indicate that it has a crucial physiologic role yet tobe explored in vivo.

IL-10R2 binding to IL-22 requires the integrity ofside chains mostly in helices A and D

IL-10R2 binds to a surface created by the inter-action of IL-22 and IL-22R.19,27 This surface is pro-posed to include a conformational change in IL-22that is induced by binding to IL-22R.27 We havedetermined that IL-22 requires the integrity of 17side chains, located in pre-helix A (A34), helix A(Y51, I52, N54, R55, T56, and K61), loop AB (A66),loop BC (V83), helix C (R88), loop CD (P113 andY114), helix D (E117, F121, L122, and L125), andhelix F (M172) (Fig. 2b and c) for optimal binding toIL-10R2. Solvent accessibility values (Table 2)indicate that most of these IL-22 side chains couldcontribute atoms to the interface with IL-10R2, as isshown in the solvent-accessible rendering of IL-22in Fig. 8a. A conformational change in IL-10 hasalready been determined to occur with binding toits high-affinity receptor, IL-10R1.39 With thisconformational change, the solvent accessibility ofthe five IL-10 side chains of helix A that contributeto the IL-10R2 interface (N39, M40, R42, S49, andR50) is altered by 9–21% in one direction or theother. Determining how significantly the putativeconformational change in IL-22 will modify thesurface displayed in Fig. 8a requires the determina-tion of an IL-22/IL-22R structure.If a conformational change in IL-22 does occur

with binding to IL-22R, then certain side-chainatoms on the surface of IL-22 may contribute to boththe IL-22R and the IL-10R2 binding sites. Yoon et al.

Fig. 7. Side-chain atoms within helix A, loop AB, and helix F of IL-22 and IL-10 define the cytokine binding interfacesfor the respective high-affinity receptor subunits. (a) A solvent-accessible surface (probe radius, 1.4 Å) rendering of aportion of IL-22's tertiary structure is shown, highlighting in red the residues that contribute to the IL-22R binding site.K161's solvent-accessible surface cannot be seen from this perspective; residues L59 and G159 are completely buried. Thenoncolored labeled residues were previously proposed, based on modeling, to participate in recognition of IL-22R;19

however, single point substitutions to alanine had no impact on binding to IL-22R in our study. (b) The same orientationas in (a) of helices A and F, and loop AB of IL-22 superimposed in stereo with the corresponding IL-10 side chains asaligned in Fig. 6. The light-yellow ribbon represents the IL-22 backbone, with the red-annotated ball-and-stick modelsrepresenting side chains that are critical for optimal binding to IL-22R, and includes those that are also important forbinding to IL-22BP (corresponding to red and blue IL-22 residues in Figs. 2b and c and 6. The orientation of the helices iscomparable to those shown in Figs. 2c, 5a, and 9. G159 of IL-22 cannot be seen well from this perspective, since it isimmediately behind K162. The gray ribbon represents the superimposed IL-10 backbone, with the blue-annotated modelsindicating side chains that are critical for IL-10R1 binding, based on the analysis of structure from IL-10/IL-10R1ECDco-crystals.38,39 Alignment, numbering, and annotation are as shown in Fig. 6. (c) A solvent-accessible surface (proberadius, 1.4 Å) rendering of IL-22 is shown, highlighting in blue and green the residues that contribute to the IL-22BPbinding site.


demonstrated, using surface plasmon resonancemethods, that R42 of IL-10 is important for bothIL-10R1 and IL-10R2 binding.39 For the interpreta-tion of our data, we assumed that an amino acidsubstitution of IL-22 that was suboptimal forbinding to IL-22R would bind similarly lesseffectively to a complex of IL-22R/IL-10R2. Sub-stitutions that had a deleterious impact on bindingto IL-22R/IL-10R2, and not to IL-22R, were

assumed to affect only IL-10R2 binding. This inter-pretation does not, however, consider those IL-22amino acids that might be involved in the bindingof both the high-affinity and the low-affinity re-ceptor subunits.We propose that D67, R73, and K162 of IL-22,

which are centrally located within the IL-22R bind-ing site and are critical for IL-22BP recognition, mayalso contribute to the IL-10R2 binding site. The

Fig. 8. Side-chain atomsmostlywithin helicesA andD of IL-22 and IL-10 define the low-affinity receptor binding sites forIL-10R2. (a)A solvent-accessible surface (probe radius, 1.4Å) rendering of a portion of IL-22's structure is shown, highlightingin purple the residues that contribute to the IL-22/IL-10R2 binding interface; T56 and L122 have very low solvent-accessiblesurfaces and cannot be seen from this perspective. While not identified as singularly critical in our study, Q48 has beenpreviously proposed to have a role in binding to IL-10R2.28 (b) The same orientation as in (a) of helices A and D of IL-22superimposed in stereo, with the corresponding IL-10 side chains as aligned in Fig. 6. The light-yellow ribbon represents theIL-22 backbone for helix A and helix D secondary structures, with the purple-annotated ball-and-stickmodels indicating sidechains that are critical for optimal binding to IL-10R2 in the presence of IL-22R. The orientation of the helices is similar to thoseshown in Fig. 5d. The gray ribbon represents the superimposed IL-10 backbone, with the green-annotatedmodels indicatingside chains that are proposed to contribute to IL-10R2 binding.39 Alignment, numbering, and annotation are as in Fig. 6.



binding of nine purified IL-22 point substitutions,originally defined as critical for optimal receptorbinding in the initial screen, was evaluated over athree-log range of concentrations. Six of thesesubstitutions bind very poorly to IL-22R at relativelylow concentrations of cytokine (i.e., 10–100 ng/ml;red and blue substitutions in Fig. 3a). However,these six substitutions do not behave comparably inthe presence of both IL-22R and IL-10R2, either in areceptor binding assay (i.e., 10–100 ng/ml; Fig. 3b)or in a cell signaling assay (0.1–1 ng/ml; Fig. 4).Three of the substitutions (V72A, I161A, and L169A;in red) bind or signal relatively well, suggesting thatthe presence of IL-10R2 shifts an equilibrium, com-pensating for an initial IL-22 defect in IL-22Rbinding. The other three substitutions (D67A,R73A, and K162A; in blue) still bind poorly in thepresence of IL-10R2, indicating that the presence ofthis subunit cannot compensate. These distinctobservations suggest that D67, R73, and K162 alsocontribute, directly or indirectly, to the interactionbetween IL-22/IL-22R and IL-10R2.To validate their structure/function model for

IL-22, Logsdon et al. evaluated the impact of 15alanine or cysteine point substitutions, demonstrat-ing that Q48, Y51, N54, and R55 in helix A (i.e., boldresidues in QQPYITNR), and Y114 and E117 inhelix D (i.e., YMQE) are important for the interac-tion between IL-22/IL-22R and IL-10R2.28 Subse-quently, Wolk et al. determined that soluble IL-10R2binds to surface-coupled IL-22 peptides that includeeither QQPYITNRTof helix A or LARLS of helix D.34

While we did not detect an impact of the Q48Asubstitution, our observations support these priorobservations and demonstrate that 12 additionalside chains of IL-22 contribute to the recognition ofIL-22/IL-22R by IL-10R2.

Does IL-10R2 recognize similar surface regionsof IL-10 and IL-22?

All cells express the IL-10R2 receptor subunit.40

This was the first clue that IL-10R2 is a receptorsubunit for several type II cytokines (i.e., IL-10, IL-22,IL-26, and IFN-λ).41 Yoon et al. determined that M40,R42, and R50 in helix A are critical for IL-10R2binding to IL-10/IL10R1, with additional side chains(i.e., N39 and S49 in helix A, and H108 and S111 inhelix D; see green bars under the IL-10 sequence inFig. 6) having subtler impacts on some assays.39 InFig. 8b are superimposed IL-22 and IL-10 helix A andhelix D structures in stereo, with the IL-22 (purple)and IL-10 (green) side chains proposed to contributeto the respective cytokine binding site for IL-10R2shown. While grossly overlapping, our systematicevaluation of mutants suggests that IL-22's bindingsite for IL-10R2 covers a broader surface than thatelucidated to date for IL-10. Alternatively, the studyof a more comprehensive panel of IL-10 substitu-tions, similar to what we have done for IL-22, mayelucidate a similar aspect of IL-10 surface contribut-ing to the IL-10R2 binding site.

IL-22 structure/function model suggests thehigh-affinity and low-affinity binding sites forother IL-10-like cytokines

Our systematic evaluation defines at the molecu-lar level those localized regions of IL-22 that arecritical for binding to IL-22R and IL-10R2, culminat-ing in signal transduction into a cell. Side chains,mostly from helices A, D, and F and loop AB, areindividually important for optimal recognition byIL-22's high-affinity and low-affinity receptor sub-units. In consideration of the actual (i.e., IL-10, IL-19,and IL-22) and modeled (IL-20, IL-24, and IL-26)structural homologies between the IL-10-like cyto-kines (summarized in Supplementary Fig. 1) andour demonstration that there is functional conserva-tion between the IL-22 and IL-10 structures (Figs. 7band 8b), we propose that positional transposition ofthe experimentally defined IL-22 receptor bindingsites is a first step towards the elucidation of otherIL-10-like cytokines' receptor binding sites. The IL-19, IL-20, IL-24, and IL-26 residues that align (seeSupplementary Fig. 1) with the IL-22R (red andblue) and IL-10R2 (yellow and green) binding sitesare shown as red (high affinity) and yellow (lowaffinity) ball-and-stick models on the ribbon struc-tures in Fig. 9. Complementary studies of thesecytokines will further determine how well structuredelineates function for the IL-10-like cytokines.

Materials and Methods

Generation and expression of IL-22 mutants

For each residue of the mature human IL-22 openreading frame, point amino acid substitutions were madeusing a three-step PCR method.42 Glycine (or silentalanine) codons were point-substituted for alanine resi-dues of IL-22, and alanine codons were point-substitutedfor all other residues. A mammalian expression plasmidthat encodes IL-22 with an N-terminal H/F tag was usedas template for the first two of three PCR steps.27 The thirdPCR generated a linear DNA expression cassette thatencoded a CMV promoter, an H/F–IL-22 protein with asingle point substitution, and an SV40 polyadenylationsequence. This panel of mutated IL-22 linear DNAexpression cassettes was used directly, without purifica-tion, for transfection of mammalian cells.43 Transfection ofsuspension HEK293FT cells (Invitrogen, Carlsbad, CA)was performed in 48-deep-well plates using 293fectin™(Invitrogen), and conditioned media were collected after5 days. For purification of select IL-22 mutants, point-substituted cDNA was subcloned into a mammalianexpression plasmid, and plasmid products used fortransient transfection of adherent HEK293. H/F-taggedand mutated IL-22 protein was purified using Ni-NTAresin (Qiagen, Valencia, CA).

IL-22 quantitation ELISA and receptor and antibodybinding ELISA

IL-22 mutant protein in conditioned media was quanti-tated by sandwich ELISA, measuring the presence of the

Fig. 9. Putative receptor binding sites for IL-19, IL-20, IL-24, and IL-26 are based on the elucidated IL-22 receptorbinding sites and the proposed conservation of the structure/function relationship among the IL-10-like cytokines. Theribbon representations of IL-19 and IL-22 backbones are from crystal structures, while those for IL-20, IL-24, and IL-26 arefrommodels generated as described in Materials andMethods. The critical IL-22 amino acid side chains that were definedas important for optimal binding to IL-22R (red), IL-10R2 (yellow), and IL-22BP (blue and green) are highlighted as ball-and-stick models and correspond to the same highlighted residues as in Figs. 2b and c and 5a–d. Based on a structuralalignment of the IL-10-like cytokines, shown at the primary sequence level in Supplementary Fig. 1, the above IL-22binding sites were transposed to the equivalent positions in the IL-19, IL-20, IL-24, and IL-26 tertiary structural renderingsshown here. We propose these to be predictive of the cognate high-affinity (red) and low-affinity (yellow) receptor subunitbinding sites for these cytokines.


N-terminal H/F-tag. Using standard methods with verygentle wash steps, H/F–IL-22 was captured with anti-FLAG HS M2 antibody covalently coated to plates (P2983;Sigma, St. Louis, MO) and detected with horseradishperoxidase (HRP)-conjugated HisProbe (Pierce, Rockford,IL). As described previously,27 ELISAs for HF–IL-22binding to receptors used plates coated indirectly witheither 25 ng/ml IL-22BP-Fc homodimers, 50 ng/ml IL-22R-Fc homodimers, or 50 ng/ml IL-22R-Fc/IL-10R2-Fcheterodimers, with the latter two containing only the ECDsof the corresponding cell surface receptor subunits.Mutantand control IL-22 cytokines in conditioned media weretested in duplicate for binding at either 200 ng/ml (IL-22R)or 20 ng/ml (IL-22R-Fc/IL-10R2-Fc and IL-22BP-Fc). RatIL22-02 and IL22-04 antibodies were coated directly at

3 μg/ml and 1 μg/ml, respectively, and IL-22 mutantswere tested in duplicate for binding at 200 ng/ml and20 ng/ml, respectively. Bound H/F–IL-22 was detected byconventional methods using HRP-conjugated HisProbe.Serial dilutions of purified IL-22mutantswere evaluated inat least two independent experiments.

IL-22-dependent BaF3 proliferation assay

A BaF3 cell line that expressed IL-10R2/YFP and IL-22R/GFP was generated by sequential retroviral trans-ductions and proliferated in response to IL-22. Serialdilutions of IL-22 (control or mutated) were added to5×103 GFP+YFP+ cells in RPMI with 10% fetal calf serum


and standard concentrations of penicillin, streptomycin,and glutamine, incubated at 37 °C and 5% CO2 for 72 h,and then evaluated for proliferation by conventionalmethods using [3H]thymidine incorporation.

IL-10 family cytokine structural and sequencealignment

A monomeric structure of IL-10 [Protein Data Bank(PDB) ID 1LK3]44 was used for superimposition with themonomeric crystal structures of IL-22 (PDB ID 1M4R)20

and IL-19 (PDB ID 1N1F),21 using the least squares methodbased on Cα positions as implemented by the Malign3Droutine ofModeler.45 Helical secondary structures for thesethree cytokines, as shown in Figs. 2 and 6 and Supple-mentary Fig. 1, were derived from Discovery StudioVisualizer 2.0 evaluation of the above structure files.Structural models for IL-20 (NP_061194.2)46 and IL-24(NP_006841.1)47 were generated using helices B through Gof IL-19 (PDB ID 1N1F) as template. Structural models forIL-26 (NP_060872.1)48 were generated using the mono-meric IL-10 structure (PDB ID 1LK3) as template. Avirtualconstruct of IL-26 containing a six-residue flexible linker(GGGSGG) inserted into the predicted DE loop betweenresidues E130 and M131 was used in model building tomimic the engineered monomeric IL-10.44 From the 100initial models for IL-20, IL-24, and IL-26, the model withthe lowest restraint violations, as defined by the molecularprobability density function, was chosen for furtheroptimization. For model optimization, an energy mini-mization cascade consisting of Steepest Descent, Conju-gate Gradient, and Adopted Basis Newton Raphsonmethods was performed until an RMS gradient of 0.01had been satisfied using CHARMm force field (AccelrysSoftware, Inc.) and Generalized Born implicit solvation, asimplemented in Discovery Studio 1.7 (Accelrys Software,Inc.). During energy minimization, backbone atom move-ments were restrained using a harmonic constraint of 10mass force.

Acknowledgements

We thank Fred Immermann and Lixin Han forguidance on statistical interpretation of data, ErikVogan and John Douhan for thoughtful reading andcritiques of the manuscript, and Dawn DeThomasfor her expertise with formatting of the figures.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.07.046

References

1. Renauld, J.-C. (2003). Class II cytokine receptors andtheir ligands: key antiviral and inflammatory modu-lators. Nat. Rev. Immunol. 3, 667–676.

2. Liang, S. C., Tan, X.-Y., Luxenberg, D. P., Karim, R.,Dunussi-Joannopoulos, K., Collins, M. & Fouser, L. A.

(2006). Interleukin (IL)-22 and IL-17 are coex-pressed by Th17 cells and cooperatively enhanceexpression of antimicrobial peptides. J. Exp. Med.203, 2271–2279.

3. Zheng, Y., Danilenko, D. M., Valdez, P., Kasman, I.,Eastham-Anderson, J., Wu, J. & Ouyang, W. (2007).Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature,445, 648–651.

4. Wolk, K., Kunz, S., Witte, E., Friedrich, M., Asadullah,K. & Sabat, R. (2004). IL-22 increases the innateimmunity of tissues. Immunity, 21, 241–254.

5. Wolk, K. & Sabat, R. (2006). Interleukin-22: a novel T-and NK-cell derived cytokine that regulates thebiology of tissue cells. Cytokine Growth Factor Rev. 17,367–380.

6. Wolk, K., Kunz, S., Asadullah, K. & Sabat, R. (2002).Cutting edge: immune cells as sources and targets ofthe IL-10 family members? J. Immunol. 168, 5397–5402.

7. Pan, H., Hong, F., Radaeva, S. & Gao, B. (2004).Hydrodynamic gene delivery of interleukin-22 pro-tects the mouse liver from concanavalin A-, carbontetrachloride-, and Fas ligand-induced injury viaactivation of STAT3. Cell. Mol. Immunol. 1, 43–49.

8. Zenewicz, L. A., Yancopoulos, G. D., Valenzuela, D. M.,Murphy, A. J., Karow, M. & Flavell, R. A. (2007).Interleukin-22 but not interleukin-17 provides protec-tion to hepatocytes during acute liver inflammation.Immunity, 27, 647–659.

9. Aujla, S. J., Chan, Y. R., Zheng, M., Fei, M., Askew, D. J.,Pociask, D. A. et al. (2008). IL-22 mediates mucosal hostdefense against Gram-negative bacterial pneumonia.Nat. Med. 14, 275–281.

10. Zheng, Y., Valdez, P. A., Danilenko, D. M., Hu, Y., Sa,S. M., Gong, Q. et al. (2008). Interleukin-22 mediatesearly host defense against attaching and effacingbacterial pathogens. Nat. Med. 14, 282–289.

11. Nickoloff, B. J. (2007). Cracking the cytokine code inpsoriasis. Nat. Med. 13, 242–244.

12. Zaba, L. C., Cardinale, I., Gilleaudeau, P., Sullivan-Whalen, M., Suarez Farinas, M., Fuentes-Duculan, J.et al. (2007). Amelioration of epidermal hyperplasiaby TNF inhibition is associated with reduced Th17responses. J. Exp. Med. 204, 3183–3194.

13. Ma, H.-l., Liang, S., Li, J., Napierata, L., Brown, T.,Benoit, S. et al. (2008). IL-22 is required for Th17 cell-mediated pathology in psoriasis-like skin inflamma-tion. J. Clin. Invest. 118, 597–607.

14. Lowes, M. A., Bowcock, A. M. & Krueger, J. G. (2007).Pathogenesis and therapy of psoriasis. Nature, 445,866–873.

15. Wolk, K., Witte, E., Wallace, E., Docke, W.-D., Kunz,S., Asadullah, K. et al. (2006). IL-22 regulates theexpression of genes responsible for antimicrobialdefense, cellular differentiation, and mobility inkeratinocytes: a potential role in psoriasis. Eur. J.Immunol. 36, 1309–1323.

16. Boniface, K., Bernard, F.-X., Garcia, M., Gurney, A.-L.,Lecron, J. C. & Morel, F. (2005). IL-22 inhibitsepidermal differentiation and induces proinflamma-tory gene expression and migration of human kerati-nocytes. J. Immunol. 174, 3695–3702.

17. Sa, S. M., Valdez, P. A., Wu, J., Jung, K., Zhong, F.,Hall, L. et al. (2007). The effects of IL-20 subfamilycytokines on reconstituted human epidermis suggestpotential roles in cutaneous innate defense andpathogenic adaptive immunity in psoriasis.J. Immunol.178, 2229–2240; [erratum appears in J. Immunol., 178(11), 7487, June 1, 2007].


18. Nagem, R. A. P., Ferreira Junior, J. R., Dumoutier, L.,Renauld, J.-C. & Polikarpov, I. (2006). Interleukin-22and its crystal structure. Vitam. Horm. 74, 77–103.

19. Logsdon, N. J., Jones, B. C., Josephson, K., Cook,J. & Walter, M. R. (2002). Comparison of inter-leukin-22 and interleukin-10 soluble receptor com-plexes. J. Interferon Cytokine Res. 22, 1099–1112.

20. Nagem, R. A. P., Colau, D., Dumoutier, L., Renauld,J.-C., Ogata, C. & Polikarpov, I. (2002). Crystal struc-ture of recombinant human interleukin-22. Structure,10, 1051–1062.

21. Chang, C., Magracheva, E., Kozlov, S., Fong, S., Tobin,G., Kotenko, S. et al. (2003). Crystal structure ofinterleukin-19 defines a new subfamily of helicalcytokines. J. Biol. Chem. 278, 3308–3313.

22. Zdanov, A. (2006). Structural studies of the inter-leukin-19 subfamily of cytokines. Vitam. Horm. 74,61–76.

23. Xie, M. H., Aggarwal, S., Ho, W. H., Foster, J.,Zhang, Z., Stinson, J. et al. (2000). Interleukin (IL)-22,a novel human cytokine that signals through theinterferon receptor-related proteins CRF2-4 and IL-22R. J. Biol. Chem. 275, 31335–31339.

24. Kotenko, S. V., Izotova, L. S., Mirochnitchenko, O. V.,Esterova, E., Dickensheets, H., Donnelly, R. P. &Pestka, S. (2001). Identification of the functionalinterleukin-22 (IL-22) receptor complex: the IL-10R2chain (IL-10Rbeta) is a common chain of both the IL-10and IL-22 (IL-10-related T cell-derived induciblefactor, IL-TIF) receptor complexes. J. Biol. Chem. 276,2725–2732.

25. Ikeuchi, H., Kuroiwa, T., Hiramatsu, N., Kaneko, Y.,Hiromura, K., Ueki, K. & Nojima, Y. (2005). Expres-sion of interleukin-22 in rheumatoid arthritis: poten-tial role as a proinflammatory cytokine. ArthritisRheum. 52, 1037–1046.

26. Andoh, A., Zhang, Z., Inatomi, O., Fujino, S.,Deguchi, Y., Araki, Y. et al. (2005). Interleukin-22, amember of the IL-10 subfamily, induces inflamma-tory responses in colonic subepithelial myofibro-blasts. Gastroenterology, 129, 969–984.

27. Li, J., Tomkinson, K. N., Tan, X.-Y., Wu, P., Yan, G.,Spaulding, V. et al. (2004). Temporal associations bet-ween interleukin 22 and the extracellular domains ofIL-22R and IL-10R2. Int. Immunopharmacol. 4, 693–708.

28. Logsdon, N. J., Jones, B. C., Allman, J. C., Izotova, L.,Schwartz, B., Pestka, S. & Walter, M. R. (2004). TheIL-10R2 binding hot spot on IL-22 is located on theN-terminal helix and is dependent on N-linkedglycosylation. J. Mol. Biol. 342, 503–514.

29. Dumoutier, L., Louahed, J. & Renauld, J. C. (2000).Cloning and characterization of IL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokinestructurally related to IL-10 and inducible by IL-9.J. Immunol. 164, 1814–1819.

30. Dumoutier, L., Van Roost, E., Colau, D. & Renauld, J.C. (2000). Human interleukin-10-related T cell-derivedinducible factor: molecular cloning and functionalcharacterization as an hepatocyte-stimulating factor.Proc. Natl Acad. Sci. U. S. A. 97, 10144–10149.

31. Lejeune, D., Dumoutier, L., Constantinescu, S., Krui-jer, W., Schuringa, J. J. & Renauld, J.-C. (2002).Interleukin-22 (IL-22) activates the JAK/STAT, ERK,JNK, and p38MAP kinase pathways in a rat hepatomacell line. Pathways that are shared with and distinctfrom IL-10. J. Biol. Chem. 277, 33676–33682.

32. Dumoutier, L., Lejeune, D., Colau, D. & Renauld, J. C.(2001). Cloning and characterization of IL-22 bindingprotein, a natural antagonist of IL-10-related T cell-

derived inducible factor/IL-22. J. Immunol. 166,7090–7095.

33. Xu, W., Presnell, S. R., Parrish-Novak, J., Kindsvogel,W., Jaspers, S., Chen, Z. et al. (2001). A soluble class IIcytokine receptor, IL-22RA2, is a naturally occurringIL-22 antagonist. Proc. Natl Acad. Sci. U. S. A. 98,9511–9516.

34. Wolk, K., Witte, E., Reineke, U., Witte, K., Friedrich,M., Sterry, W. et al. (2005). Is there an interactionbetween interleukin-10 and interleukin-22? GenesImmun. 6, 8–18.

35. Xu, T., Logsdon, N. J. & Walter, M. R. (2005). Structureof insect-cell-derived IL-22. Acta Crystallogr. Sect. D,61, 942–950.

36. Langer, J. A., Cutrone, E. C. & Kotenko, S. (2004). TheClass II cytokine receptor (CRF2) family: overviewand patterns of receptor–ligand interactions. CytokineGrowth Factor Rev. 15, 33–48.

37. de Oliveira Neto, M., Ferreira, J. J., Colau, D., Fischer,H., Nascimento, A., Craievich, A. et al. (2008).Interleukin-22 forms dimers that are recognized bytwo interleukin-22R1 receptor chains. Biophys. J. 94,1754–1765.

38. Josephson, K., Logsdon, N. J. & Walter, M. R. (2001).Crystal structure of the IL-10/IL-10R1 complexreveals a shared receptor binding site. Immunity, 15,35–46.

39. Yoon, S. I., Logsdon, N. J., Sheikh, F., Donnelly, R. P. &Walter, M. R. (2006). Conformational changes mediateinterleukin-10 receptor 2 (IL-10R2) binding to IL-10and assembly of the signaling complex. J. Biol. Chem.281, 35088–35096.

40. Kotenko, S. V. (2002). The family of IL-10-relatedcytokines and their receptors: related, but to whatextent? Cytokine Growth Factor Rev. 13, 223–240.

41. Kotenko, S. V. & Langer, J. A. (2004). Full house: 12receptors for 27 cytokines. Int. Immunopharmacol. 4,593–608.

42. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. &Pease, L. R. (1989). Site-directed mutagenesis byoverlap extension using the polymerase chain reac-tion. Gene, 77, 51–59.

43. Liang, X., Teng, A., Braun, D. M., Felgner, J., Wang, Y.,Baker, S. I. et al. (2002). Transcriptionally activepolymerase chain reaction (TAP): high throughputgene expression using genome sequence data. J. Biol.Chem. 277, 3593–3598.

44. Josephson, K., Jones, B. C., Walter, L. J., DiGiacomo,R., Indelicato, S. R. & Walter, M. R. (2002). Non-competitive antibody neutralization of IL-10 revealedby protein engineering and X-ray crystallography.Structure, 10, 981–987.

45. Marti-Renom, M. A., Stuart, A. C., Fiser, A., Sanchez,R., Melo, F. & Sali, A. (2000). Comparative proteinstructure modeling of genes and genomes. Annu. Rev.Biophys. Biomol. Struct. 29, 291–325.

46. Blumberg, H., Conklin, D., Xu, W. F., Grossmann, A.,Brender, T., Carollo, S. et al. (2001). Interleukin 20:discovery, receptor identification, and role in epider-mal function. Cell, 104, 9–19.

47. Jiang, H., Lin, J. J., Su, Z. Z., Goldstein, N. I. & Fisher,P. B. (1995). Subtraction hybridization identifies a novelmelanoma differentiation associated gene, mda-7,modulated during human melanoma differentiation,growth and progression. Oncogene, 11, 2477–2486.

48. Knappe, A., Hor, S., Wittmann, S. & Fickenscher, H.(2000). Induction of a novel cellular homolog ofinterleukin-10, AK155, by transformation of T lympho-cytes with herpesvirus saimiri. J. Virol. 74, 3881–3887.

223 IL22R, IL10R2 and IL22BP binding sites are topologically juxtaposed on adjacent and overlapping...

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