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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Structure. Author manuscript; available in PMC Sep 10, 2009.
Published in final edited form as:
PMCID: PMC2637415
NIHMSID: NIHMS69687
Structure of IL-22 bound to its high affinity IL-22R1 chain
Brandi C. Jones,1 Naomi J. Logsdon,1 and Mark R. Walter1,2
1Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham, Birmingham, Alabama 35294
2Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294
4Correspondence should be addressed to Mark R.Walter, Phone (205) 934-9279, FAX (205) 934-0480, Email: walter/at/uab.edu
IL-22 is an IL-10 family cytokine that initiates innate immune responses against bacterial pathogens and contributes to immune disease. IL-22 biological activity is initiated by binding to a cell surface complex composed IL-22R1 and IL-10R2 receptor chains and further regulated by interactions with a soluble binding protein, IL-22BP, which shares sequence similarity with extracellular region of IL-22R1 (sIL-22R1). IL-22R1 also pairs with the IL-20R2 chain to induce IL-20 and IL-24 signaling. To define the molecular basis of these diverse interactions, we have determined structure of the IL-22/sIL-22R1 complex. The structure, combined with homology modeling and surface plasmon resonance studies, define the molecular basis for the distinct affinities and specificities of IL-22 and IL-10 receptor chains that regulate cellular targeting and signal transduction to elicit effective immune responses.
Keywords: IL-10 family, IL-22, IL-22R1, IL-10R2, specificity, promiscuity
Interleukin-22 (IL-22) is an α-helical cytokine produced by activated DC and T cells, including the recently identified Th17 lineage (Dumoutier et al., 2000a; Liang et al., 2006). Despite its production by immune cells, IL-22 biological activity is specifically targeted to cells of epithelial origins where it induces pro-inflammatory activities that include the production of early systemically circulated defense proteins (acute phase proteins) (Dumoutier et al., 2000b). More recently, IL-22 has been shown to induce anti-microbial proteins, including S100s and defensins, in colonic and lung epithelial cells, which are critical for host defense against enteric and pulmonary bacterial pathogens (Aujla et al., 2008; Liang et al., 2006; Wolk et al., 2004; Zheng et al., 2008). This suggests IL22 allows coordination between the adaptive and innate immune systems to fight invading pathogens.
In addition to its pro-inflammatory activities, IL-22 confers a protective effect on the liver by promoting hepatocyte survival (Radaeva et al., 2004; Zenewicz et al., 2007). IL-22 also enhances lung epithelial cell proliferation and the maintenance of trans-epithelial resistance to injury (Aujla et al., 2008). IL-22-mediated protective effects have also been observed in the gut, where IL-22 knockout mice, subjected to C. rodentium infection, exhibit increased epithelial cell damage and increased mortality (Zheng et al., 2008). Thus, IL-22 exhibits paradoxical pro- and anti- inflammatory properties on epithelial cells, which are strikingly similar to the activity profile of IL-10 on immune cells (Moore et al., 2001).
IL-22 and IL-10 induce cellular responses by engaging heterodimeric cell surface receptor complexes that contain IL-22R1 and IL-10R1 chains, respectively, and the shared signal transducing receptor, IL-10R2 (Figure 1, (Kotenko et al., 2001a; Xie et al., 2000). Formation of the IL-22/IL-22R1/IL-10R2 complex activates intracellular kinases (JAK1, Tyk2, and MAP kinases) and transcription factors, especially STAT3, leading to IL-22 cellular responses (Lejeune et al., 2002). IL-10/IL-10R1/IL-10R2 assembly induces overlapping kinases and also predominantly activates STAT3 (Moore et al., 2001).
Figure 1
Figure 1
Schematic diagram of protein complexes formed by IL-22 receptors
IL-22R1 is selectively expressed on skin and epithelial cells, while IL-10R1 is predominantly expressed on DC, monocytes, and macrophages (Nagalakshmi et al., 2004). In contrast, IL-10R2 is expressed on all cell types, which reflects its role as a shared receptor in IL-22, IL-10, IL-26, IL-28, and IL-29 receptor complexes. Surface plasmon resonance (SPR) studies are consistent with the targeting function of IL-22R1 and IL-10R1 (Logsdon et al., 2004; Logsdon et al., 2002; Yoon et al., 2005; Yoon et al., 2006). sIL-22R1 and sIL-10R1 both exhibit high affinity (~nM) for their respective ligands. In contrast, IL-10R2 interactions are very weak (~µM-mM), which may facilitate promiscuous, but essential, interactions in multiple cytokine complexes resulting in cellular signaling responses.
IL-22 and IL-10 are structurally similar to each other and the rest of the IL-10 cytokine family, which includes IL-19, IL-20, IL-24, and IL-26 (Pestka et al., 2004). In addition to specific high affinity interactions with IL-22, IL-22R1 also pairs with IL-20R2 to induce IL-20 and IL-24 signaling (Dumoutier et al., 2001a)(Figure 1). Like IL-22, IL-20 and IL-24 have been implicated skin homeostasis and pathology (Blumberg et al., 2001; Kunz et al., 2006; Sa et al., 2007). IL-20 induces keratinocyte proliferation and over expression in mice leads to a psoriasis-like phenotype (Blumberg et al., 2001). In contrast to IL-22, IL-20 and IL-24 are expressed predominantly by monocytes and keratinocytes. Under Th1 conditions, IL-22R1 and IL-20R2 chains are also expressed on keratinocytes (Kunz et al., 2006). This suggests IL-20 and IL-24, via the IL-22R1/IL-20R2 complex, may provide a communication link between monocytes and keratinocytes, as well as a feedback loop on keratinocytes themselves.
The high level of IL-22 in human psoratic plaques and its pathogenic role in a mouse model of psoriasis, suggests antagonists that block the IL-22/sIL-22R1 interaction may have therapeutic potential (Boniface et al., 2007; Ma et al., 2008; Wolk et al., 2006). Furthermore, cells tightly regulate IL-22 activities by expressing an IL-22 binding protein (IL-22BP), which shares 34% sequence identity with the extracellular region of IL-22R1 (sIL-22R1) (Dumoutier et al., 2001b; Kotenko et al., 2001b; Wei et al., 2003; Xu et al., 2001). Three splice forms of human IL-22BP have been identified in human cells (Kotenko et al., 2001b). Mature IL-22BP isoform-2 (IL-22BPI2, swiss prot id Q969J5-2) is 210 amino acids long and predicted to adopt a structure similar to sIL-22R1, while isoform-3 (IL-22BPI3, swiss prot id Q969J5-3) is 109 amino acids long and should adopt a structure similar to the sIL-22R1 D1 domain (Figure 1). The biochemical properties and functional roles of the IL-22BP isoforms have not been elucidated.
The fidelity of intracellular signaling between innate and adaptive immune systems is critical to balance pathogen elimination versus damage to host tissues. IL-22 and IL-22R1 play critical roles in this process. Not only by activation of the IL-22/IL-22R1/IL-10R2 complex, but through additional diverse interactions between IL-22 / IL-22BP and the participation of IL-22R1 in IL-20/IL-22R1/IL-20R2 and IL-24/IL-22R1/IL-20R2 complexes. To understand the molecular basis of these protein-protein interactions, we have determined the structure of the IL-22/sIL-22R1 complex. Comparison of IL-22/sIL-22R1 and IL-10/sIL-10R1 structures, combined with homology modeling and SPR analysis of IL-22/IL-22BP and IL-22/IL-22R1/IL-10R2 complexes, provides a molecular understanding of how these molecules form specific and promiscuous complexes with affinities that differ by ~6 orders of magnitude. (Table 1, Figure 1).
Table 1
Table 1
Binding Constants for IL-22 and IL-10
Structure Determination
The IL-22/sIL-22R1 structure was solved by SAD phasing and refined at 2.5Å resolution to Rcryst and Rfree values of 22.2% and 27.0%, respectively (Table S1). IL-22 used for crystallization (IL-22N-QQ) contained no N-linked glycosylation and sIL-22R1 (sIL-22R1DDQ,) contains carbohydrate at one of three sites, Asn-172sIL-22R1 (Jones et al., 2008), which is observed in the structure (Figure 2). Asparagines Asn-68IL-22 and Asn-97IL-22 were mutated to glutamine to make IL-22N-QQ, while asparagines Asn-80sIL-22R1 and Asn-87sIL-22R1 were replaced by aspartic acids and Thr-89sIL-22R1 was replaced by glutamine to make sIL-22R1DDQ. IL-22N-QQ and sIL-22R1DDQ exhibit essentially the same affinity for one another as their glycosylated counterparts (Table 1), suggesting the IL-22N-QQ/sIL-22R1DDQ crystal structure (IL-22/sIL-22R1 throughout the rest of text) accurately reflects the cell surface IL-22/IL-22R1 complex.
Figure 2
Figure 2
Structure of the IL-22/IL-22R1 complex
Overall structure of IL-22/sIL-22R1
IL-22 and sIL-22R1 form a 1:1 complex in the crystal (Figure 2). sIL-22R1 adopts an “L” shaped structure orienting its two fibronectin type III (FBNIII) domains (D1 and D2) at approximately right angles to one another. The structure of sIL-22R1 is similar to sIL-10R1 (Josephson et al., 2001), except the D2 domain of sIL-22R1 is rotated by ~20°, relative to sIL-10R1, about an axis parallel to the D2 β-strands. Although considerable conformational differences are observed among free IL-22 structures (Nagem et al., 2002; Xu et al., 2005), sIL-22R1 bound IL-22 molecules are essentially identical (r.m.s.d. = 0.36Å). The IL-22/sIL-22R1 complex is most similar to one half of the dimeric IL-10/sIL-10R1 complex (Josephson et al., 2001). Upon superposition of IL-22 onto one subunit of the IL-10 dimer (2.7Å r.m.s.d, 109 atoms), the C-termini of sIL-22R1 and sIL-10R1 chains, prior to their entry into the membrane, are separated by ~12Å (Figure 2C). Despite similar architectures, IL-22/sIL-22R1 does not form an “IL-10 like” dimeric complex in the crystal lattice.
The IL-22/sIL-22R1 interface
The IL-22/sIL-22R1 interface consists of five sIL-22R1 loops (L2-L6) located at the interface of the D1 and D2 domains, which engage IL-22 residues presented on helix A, the AB loop, and helix F (Figure 2, Figure 3). The interface may be separated into two major contact surfaces, site 1a and site 1b. L2-L4 loops within the D1 domain form contacts in site 1a, while loops L5 and L6, from D2, contact site 1b.
Figure 3
Figure 3
IL-22 and IL-10 exhibit distinct site 1 structures and binding surfaces
Site 1a contributes ~72% of the total buried surface area and 11 of 12 hydrogen bond/salt bridge interactions in the IL-22/sIL-22R1 complex (Figure 3, Figure 4, Table 2). The predominant feature of site 1a is the insertion of Tyr-60sIL-22R1 and Gly-61sIL-22R1 into a small cavity on IL-22 created at the intersection of the helix F and the AB loop (Figure 2B). The base of the cavity is formed by helix F residues Lys-162IL-22 and Glu-166IL-22, which each form hydrogen bonds to the OH atom of Tyr60sIL-22R1. Gly61sIL22R1 also forms a hydrogen bond with the O atom of Thr72IL-22 located in the AB loop. The contacts formed by Tyr60sIL-22R1 and Gly61sIL-22R1 are identical to those previously described in the IL-10/sIL-10R1 complex for residues Tyr-43sIL-10R1, Gly-44sIL-10R1, Asp44IL-10, Lys-138IL-10, Glu-142IL-10. Thus, the major site 1a binding epitope is conserved in IL-22/sIL-22R1 and IL-10/sIL-10R1 complexes (Figure 4).
Figure 4
Figure 4
Comparison of IL-22/sIL-22R1 and IL-10/sIL-10R1 interfaces
Table 2
Table 2
Potential hydrogen bonds in IL-22/IL-22R1 and IL-22/IL-22BP complexes
In contrast to the extensive site 1a interface, IL-22/sIL-22R1 site 1b is very small and distinct from IL-10/sIL-10R1 (Figure 2, Figure 4). It is made up almost entirely of Van der Waals interactions between IL-22 helix F residues, Met-172IL-22 and Arg-175IL-22, and sIL-22R1 L6 residues, Thr-207sIL-22R1 and Trp-208sIL-22R1 (Figure 2B, ,4E).4E). Site 1b contains one salt bridge between Lys-44IL-22, located in the preA helix, and Asp-162sIL-22R1 located on the L5 loop.
Mechanisms regulating IL-22/sIL-22R1 and IL-10/sIL-10R1 affinity
Using the structures of IL-22/sIL-22R1 and IL-10/sIL-10R1, we sought to explain the distinct binding kinetics of the complexes (Table 1). Our past SPR analysis of IL-22/sIL-22R1 interactions used amine coupled IL-22, which can prevent the accurate estimation of on-rates (Logsdon et al., 2004; Logsdon et al., 2002). To test for this possibility, we re-designed our SPR experiment by injecting IL-22 over sIL-22R1-his6 captured on an anti-His6 antibody surface. Using this experimental setup, an on-rate of 1.3 × 106 M−1sec−1 was obtained for the IL-22/sIL-22R1 interaction, which is an ~9 fold increase over previous amine coupled experiments and ~sixfold faster than observed for the IL-10/sIL-10R1 interaction (Table 1). Interestingly, the overall affinity of each complex is similar (1nM versus 0.47nM), but the occupancy time of the ligand on the receptor, quantified by the complex half-life (T1/2) is 7.4 minutes for IL-22/sIL-22R1 versus 44 minutes for IL-10/sIL-10R1.
The amounts of buried surface area and specific contacts in the IL-22/IL-22R1 and IL-10/sIL-10R1 complexes are consistent with the observed binding kinetics. The IL-22/sIL-22R1 interface buries 1,585A2 of surface compared to 1,893A2 for IL-10/sIL-10R1. In both complexes, site 1a forms the predominant contact surface. In IL-22/sIL-22R1, it consists of 1,141A2 of buried surface area and 11 of 12 hydrogen bonds identified in the complex (Table 2). Similarly, the IL-10/sIL-10R1 site 1a interface consists of 1,249A2 (9% larger than IL-22/sIL-22R1) and forms 10 hydrogen bonds (Yoon et al., 2005).
Larger differences are observed in site 1b, which consists of 444Å2 in IL-22/sIL-22R1 compared to 664A in IL-10/sIL-10R1 (a 33% difference). In addition, IL-10/sIL-10R1 site 1b contains 6 hydrogen bonds compared to only one in IL-22/sIL-22R1 (Figure 4E, F). As a result, the number of hydrogen bonds per 100A2 of surface area (HB/100A) is ~1.7 throughout the entire IL-10/sIL-10R1 site 1 interface. In contrast, IL-22/sIL-22R1 site 1a and 1b interfaces exhibit HB/100A values of 1.9 and 0.5, respectively. These comparisons suggest the reduced size (buried surface area and contacts) of IL-22/sIL-22R1 site 1b is mainly responsible for its faster off-rate compared to IL-10/sIL-10R1.
IL-22/IL-22BP interactions
To further evaluate the role of site 1a and 1b in IL-22 binding affinity, we studied the binding kinetics of IL-22BP isoforms IL-22BPI2 and IL-22BPI3 for IL-22 (Figure 5, Table 1). Homology models of IL-22/IL-22BPI2 and IL-22/IL-22BPI3 reveal IL-22BPI2 forms site 1a and 1b contacts with IL-22, while IL-22BPI3 only forms site 1a contacts. With one exception, the models suggest all site 1a IL-22/IL-22R1 contacts are conserved in IL-22/IL-22BPI2 and IL-22/IL-22BPI3 complexes (Table 2). Thus, the IL-22/IL-22BPI3 complex essentially mimics an IL-22/sIL-22R1 site 1a interface. Using SPR methods, IL-22/IL-22BPI3 exhibits a dissociation constant (Kd) of ~4nM compared to 1nM for the IL-22/sIL-22R1 complex (Figure 5, Table 1). The off-rate of the IL-22/IL-22BPI3 complex (2.4 × 10−3M−1sec−1) is only 1.5 fold faster than observed for the IL-22/sIL-22R1 complex. Thus, both the structure analysis (Figure 4E) and SPR data suggest site 1b contributes very little to the overall stability of the IL-22/sIL-22R1 complex.
Figure 5
Figure 5
Binding and structure of IL-22BP isoforms
In contrast to sIL-22R1 and IL-22BPI3, homology modeling of the IL-22/IL-22BPI2 complex suggests the D2 domain of IL-22BPI2 forms extensive site 1b contacts with IL-22 (Figure 5D). Key features of this interface are 5 new salt-bridge/hydrogen bonds (Table 2), and the formation of a hydrophobic cluster comprised of Trp-116IL-22BPI2 and Leu-208IL-22BPI2 and IL-22 residues Phe57IL-22, Phe171IL-22, and Met-172IL-22.
Consistent with the homology model, SPR analysis revealed a Kd of ~1pM, with a T1/2 of 4.7 days, for the IL-22/IL-22BPI2 interaction (Table 1). The extremely slow off-rate of the IL-22/IL-22BPI2 complex was determined by a 22 hour SPR experiment that monitored the dissociation of IL-22BPI2 from amine coupled IL-22 surfaces (Figure 5). Since Asn-54IL-22 makes a putative hydrogen bond with Asp-209IL-22BPI2, we characterized the loss of IL-22BPI2 from amine coupled surfaces of glycosylated IL-22 (IL-22NNN), as well as IL-22 mutants where N-linked carbohydrate attachment residues Asn-54IL-22(IL-22QNN) and Asn-68IL-22(IL-22NQN) were changed to glutamines. Consistent with our model, a total of 8RU of IL-22BPI2 dissociated from amine coupled IL-22NNN and IL-22NQN in the 22 hour period, while 28RU of IL-22BPI2 dissociated from the IL-22QNN surface, presumably by disrupting the Asn-54IL-22/Asp-209IL-22BPI2 interaction by the glutamine mutation. These studies further infer a link between high affinity binding of IL-22/IL-22BPI2 and IL-10/sIL-10R1 with the size of site 1b.
Specificity of IL-22/sIL-22R1 and IL-10/IL-10R1 complexes
IL-22 and IL-10 cellular responses require efficient targeting to specific cells via their high affinity chains, IL-22R1 and IL-10R1. Closer structural analysis reveals the conserved site 1a L2/AB loop recognition motif, which consists of Tyr-60sIL-22R1 and Gly-61sIL-22R1 is a critical specificity determinant (Figure 4). This is because the precise geometry of the hydrogen bonds formed by these residues cannot be accurately reproduced in non-cognate complexes (e.g. IL-22/sIL-10R1 or IL-10/sIL-22R1) without steric clashes between other regions of the molecules (Figure 2C).
For example, superposition of IL-22 onto the IL-10/sIL-10R1 results in close contacts (~1A) between mainchain atoms from the IL-22 pre-A helix and sIL-10R1 L6 loop, which would prevent complex formation. Likewise, when IL-10 is superimposed onto the IL-22/sIL-22R1 complex, close contacts occur between the AB loop of IL-10 and the L2 loop of sIL-22R1. Proper hydrogen bonding between the IL-10 AB loop and sIL-22R1 L2 loop can be obtained by reorienting IL-10 on the surface of IL-22R1. However, this results in additional disruptions in site 1b located on the other side of the complex. Thus, the requirement for specific contacts in two spatially distinct regions of the interfaces, site 1a and site 1b, provides critical constraints that ensure specificity between IL-22/IL-22R1 and IL-10/IL-10R1 complexes.
Although essentially the same residues form IL-22/sIL-22R1 and IL-10/IL-10R1 site 1 interfaces, the amount of buried surface area and the number of contacts contributed by each residue is very different (Figure 3). For example, IL-22 AB loop residues Thr70IL-22 (108Å2), Asp71IL-22 (74Å2), and Arg73IL-22 (102Å2) bury the most (>70Å2) surface area in the IL-22/sIL-22R1 complex. Asp44IL-10, which is structurally identical to Thr70IL-22, buries the most surface area (116Å2) of any residue in the IL-10/sIL-10R1 complex. However, IL-10 helix A residues bury large amounts of surface area (Pro20IL-10, 71Å2; Arg24IL-10, 87Å2; Arg27IL-10, 66Å2; Lys34IL-10, 69Å2; Gln38IL-10, 73Å2) into sIL-10R1, which is not observed in IL-22/sIL-22R1 (Figure 3).
Because of the extensive interactions made between the IL-22 AB loop and sIL-22R1, including 5 of 12 hydrogen bonds in the interface (Table 2, Figure 3), the AB loop sequences of other IL-10 family members were compared to determine if these residues were conserved in IL-20 and IL-24, which also bind to IL-22R1. As shown in Figure 4D, IL-20 and IL-24, but not IL-10, IL-19, or IL-26 contain two of the three IL-22 AB loop residues (IL-20, Arg-73IL-22 and Asp-71IL-22; IL-24, Arg-73IL-22 and Thr-70IL-22) suggesting they are important specificity determinants that promote interactions with IL-22R1.
Promiscuous IL-10R2 binding to IL-22/IL-22R1 and IL-10/IL-10R1 complexes
IL-22 and IL-10 both engage the common low affinity signal transducing chain, IL-10R2 (Kotenko et al., 2001a; Kotenko et al., 1997; Xie et al., 2000). A previous model of the IL-10/IL-10R1/IL-10R2 ternary complex, generated without any biochemical constraints, defined the general location of the IL-10R2 binding site (Pletnev et al., 2005). Here, our goal was to generate models of the IL-22 and IL-10 ternary signaling complexes (TC) that explain the distinct affinities of sIL-10R2 for IL-22/sIL-22R1 (Kd ~14µM) and IL-10/sIL-10R1 (Kd ~234µM) binary complexes (Table 1). To accomplish this, we used IL-22/sIL-22R1 and IL-10/sIL-10R1 (Josephson et al., 2001) structures and mutagenesis studies, that mapped the IL-10R2 binding sites on IL-10 (Yoon et al., 2006) and on IL-22 (Logsdon et al., 2004), as experimental constraints for TC model generation.
As shown in Figure 6, sIL-10R2 contacts IL-22/sIL-22R1 and IL-10/sIL-10R1 binary complexes at three distinct surfaces labeled site 2a, 2b, and 2c. Site 2a and 2b are predominantly between sIL-10R2 and helices A and D of IL-22 and IL-10, while receptor D2sIL-10R2-DsIL-22R1 or D2sIL-10R2-D2sIL-10R1 interactions make up site 2c. A total of 10 hydrogen bond/salt bridge interactions were identified in the IL-22 and IL-10 site 2 interfaces (Table S2). Five of the 10 interactions use the same sIL-10R2 residue in both IL-22 and IL-10 TC complexes. The amount of surface area buried by IL-10R2 in the IL-10 TC site 2 interface is ~1200A2, which corresponds to a HB/100A ratio of ~0.8, which is similar to the IL-22/sIL-22R1 site 1b interface (0.5 HB/100A, Figure 4E). The IL-22 TC site 2 interface is smaller (~900A2) resulting in a slightly larger HB/100A ratio of ~1.1.
Figure 6
Figure 6
Models of the IL-22/IL-22R1/IL-10R2 and IL-10/IL-10R1/IL-10R2 ternary complexes
In addition to characterizing the low affinity binding properties of site 2, the TC models suggest structural differences in the ligands determines IL-10R2 affinity for IL-22 and IL-10 binary complexes. In the IL-22 TC, the N-terminal end of helix A presents a knob-like surface for interactions with the binding loops of sIL-10R2 (Figure 2E). In contrast, the surface presented by helix A in IL-10 is essentially flat, which is consistent with its lower affinity for IL-10R2. Arg-55IL-22 and Arg-32IL-10 both form conserved salt bridge interactions with Asp-84sIL-10R2, but structural differences that could alter sIL-10R2 affinity are present. Specifically, the sidechain conformation of Arg-55IL-22 is fixed by additional contacts with Tyr-51IL-22, while there are no such constraints on Arg-32IL-10. These findings are consistent with the 120µM affinity of the IL-22/IL-10R2 interaction compared to the ~mM affinity of the IL-10/IL-10R2 interaction (Table 1).
The most extensive contacts in the IL-10 TC are observed in site 2b, which consists of the tripartite interaction between Arg-24IL-10, the L6 loop of sIL-10R1, and Asn-147sIL-10R2. In a manner similar to the presentation of Arg-55IL-22 on the knob of IL-22, sIL-10R1 interactions with Arg-24IL-10 orient the sidechain for interactions with Asn-147sIL-10R2. The most diverse contacts in the IL-22 and IL-10 TCs occur in site 2c. In particular, Glu-121sIL-10R2 forms a hydrogen bond with Ser-174sIL-10R1 in the IL-10 TC. However, in the IL-22 TC, Glu-121sIL-10R2 interacts with the N-acetylglucosamine (NAG) attached to Asn-174sIL-22R1 (Figure 2D, 2E, Figure 6). This is especially interesting because previous characterization of IL-22/sIL-10R2 interactions found carbohydrate attached to Asn-54IL-22 to be important for sIL-10R2 binding (Logsdon et al., 2004).
IL-22 folds into a compact monomeric structure, in contrast to IL-10, which assembles into domain-swapped dimers (Nagem et al., 2002; Walter and Nagabhushan, 1995; Xu et al., 2005; Zdanov et al., 1995). Interestingly, recent X-ray scattering data reveals IL-22 forms non-covalent dimers, and tetramers, in solution that adopt “V-shaped” structures similar to the IL-10 dimer (de Oliveira Neto et al., 2007). Although we do not observe an IL-10-like dimer complex in the crystal lattice, it is clear that IL-22 and IL-10 form receptor complexes with very similar architectures. This suggests the overall receptor complex structure, with the exception of oligomerization (.e.g. monomer-dimer transition), is not responsible for differences in IL-22 and IL-10 signaling.
FRET data has revealed IL-10R1 and IL-10R2 associate with one another on the cell surface in the absence of IL-10 (Krause et al., 2006). Since IL-22R1 pairs with IL-10R2 to induce IL-22 signaling, IL-22R1 and IL-10R2 are also predicted to form cell surface heterodimers (Krause et al., 2006). Based on mechanistic studies of growth hormone and erythropoietin signaling, binding of IL-22 and IL-10 to their respective R1/R2 heterodimers is expected to induce rotational and/or conformational changes in the receptors leading to cell signaling (Brown et al., 2005; Remy et al., 1999). Although many of the general features of the IL-22 and IL-10 signaling complexes are conserved, IL-22 and IL-10 exhibit unique specificities and receptor binding kinetics, which are dependent on the structural details of the site 1 and site 2 interfaces. IL-22/IL-22R1 and IL-10/IL-10R1 site 1 contacts exhibit Kd values (nM) that are at least 4 orders of magnitude higher than site 2 contacts made by the common IL-10R2 chain (µM).
Because of the large differences in the site 1 and site 2 binding affinity/kinetics, IL-22 and IL-10 TC activation are “R1 dominant”. This suggests that despite the pre-association of R1 and R2 chains on the cell surface, cell specificity/targeting, and receptor occupancy time are controlled by the binding kinetics of the R1 chains, which are selectively expressed on different cell types (Nagalakshmi et al., 2004). In contrast to IL-22R1 and IL-10R1, IL-10R2 is ubiquitously expressed on all cell types (Nagalakshmi et al., 2004). The weak binding of IL-10R2, via site 2, suggests it functions as a “sensor chain”, which activates signaling based on the kinetics of the R1 chain, via site 1. Weak site 2 binding also promotes promiscuous IL-10R2 interactions with IL-22, IL-10, and other cytokines (IL-26, IL-28, and IL-29), and reduces the number of unique chains required for IL-10 family cytokine signaling. Our conclusions, based on SPR data, agree with IL-10 cell surface binding studies that reveal IL-10 binds to cells expressing only the IL-10R1 chain with an identical affinity as cells expressing both IL-10R1 and IL-10R2 (Ding et al., 2001).
Additional regulation of IL-22 cell targeting/signaling is suggested by the structure and binding properties of IL-22BP isoforms. Our analysis suggests IL-22/IL-22BPI2 forms an extremely tight (Kd ~1pM) interaction with IL-22, which differs considerably from the 1nM Kd obtained by another recent SPR analysis of the IL-22/IL-22BPI2 complex (Wolk et al., 2007). Possible reasons for these discrepancies are the use of amine coupled IL-22 by Wolk et al., which can lead to reduced on-rates and the use of short dissociation times, which can under estimate slow off-rates. Also in contrast to Wolk et al., our capture experiments suggest sIL-22R1 and IL-22BPI2 exhibit essentially identical on-rates. However, the off-rates differ dramatically resulting in T1/2 values of ~7 minutes for IL-22/sIL-22R1 compared to ~4.7 days for IL-22/IL-22BP. The impact of these findings on IL-22 biology depends on IL-22, IL-22R1 and IL-22BP levels in various tissues, which are now being explored in more detail.
SPR analysis of IL-22BPI3 reveals it displays binding kinetics similar to the IL-22/sIL-22R1 complex. Analysis of IL-22BPI3 confirms the importance of site 1a for IL-22 binding and the small role of site 1b in IL-22/sIL-22R1 interactions. The functional role of the IL-22BPI3 isoforms remains unclear. Relative to IL-22BPI2, IL-22BPI3 is essentially inactive due to a ~2800 fold reduction in IL-22 affinity, which suggests splicing may be a mechanism to inactivate IL-22BPI2. However, since IL-22BPI3 does retain “sIL-22R1-like” affinity for IL-22, it is formally possible that IL-22/IL-22BPI3 complexes have a yet unknown role in IL-22 biology.
In addition to providing a structural model that explains observed IL-10R2 affinities and promiscuity, the IL-22 TC model has also revealed a possible role for N-linked glycosylation in IL-22R1/IL-10R2 site 2c interactions. This is interesting since carbohydrate attached to IL-22 (Asn54IL-22) was previous shown to be important for IL-10R2 binding (Logsdon et al., 2004). Docking studies were performed in the presence of Asn-54IL-22-linked carbohydrate obtained from the crystal structure of glycosylated IL-22 (Xu et al., 2005). Thus, the IL-22 TC contains sufficient space to accommodate N-linked carbohydrate between sIL-22R1 and sIL-10R2, but an explanation for how mutation of Asn-54IL-22 to glutamine disrupts sIL-10R2 binding and cell signaling could not be identified (Logsdon et al., 2004). This may be due to errors in the modeled sIL-10R2 loops, or Asn-54IL-22 may play an indirect role (e.g. mediate conformational changes) in activating the IL-22 TC.
In addition to IL-22 signaling complexes, IL-22R1 along with IL-20R2, also form promiscuous IL-20 and IL-24 TCs. However, IL-20/sIL-22R1 interactions are weak, based on our inability to isolate a stable IL-20/sIL-22R1 complex by size exclusion chromatography (N.J. Logsdon and M.R Walter, unpublished results). Thus, in contrast to the “R1 dominant” IL-22 and IL-10 signaling complexes, sIL-22R1 appears to play a different role in IL-20 and IL-24 complexes. We have identified two critical recognition elements that facilitate promiscuous binding by IL-22R1. First, IL-20 and IL-24 conserve two of three IL-22 AB loop residues (Thr-70IL-22, Asp-71IL-22, and Arg-73IL-22) that form extensive contacts with sIL-22R1. Second, the low affinity of the IL-20/sIL-22R1 is consistent with the small site 1b interface observed in IL-22/sIL-22R1, which in our specificity model, is critical for promiscuous contacts. The necessity of these affinity and specificity differences may allow fine tuning of IL-19, IL-20, and IL-24 signaling via IL-22R1/IL-20R1 versus IL-20R1/IL-20R2 complexes in the skin compared to other tissues. The detailed structural basis that regulates these distinct signaling mechanisms will be the focus of future investigations.
Crystallization, data collection, and structure determination
Crystals of the IL-22/sIL-22R1 complex were obtained as described by Jones et al. (Jones et al., 2008). X-ray diffraction data were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois. The structure of the IL-22/sIL-22R1 complex was solved by molecular replacement, using MOLREP (CCP4,(Collaborative Computational Project 4, 1994)), and SAD phasing methods implemented in CNS version 1.1 (Brunger et al., 1998). The sIL-22R1 model was built using O (Jones et al., 1991). Buried surface area was calculated using NACCESS (Hubbard and Thorton, 1993). Structural alignments were performed using STAMP (Russell and Barton, 1992) implemented in VMD (Humphrey et al., 1996). Figures were made using the ribbons and Pymol (Carson, 1997; DeLano, 2002).
Modeling
IL-22BP and IL-10R2 models were made using MODELER v8.1. (Eswar et al., 2003). sIL-22R1 and IL-10R1 were used as the structural templates for the IL-22BPs and IL-10R2 model generation, respectively, using the sequence alignments shown in Figure 5 and Figure S2. sIL-10R2 docking was performed manually using pymol (DeLano, 2002) such that IL-10R2 exhibited essentially identical positions in IL-22 and IL-10 TC complexes and was consistent with major finding of the mutagenesis data (Logsdon et al., 2004; Yoon et al., 2006).
SPR analysis
SPR experiments were performed on a BIAcore 2000 system at 20°C using a running buffer containing 10mM Hepes, pH 7.4, 0.15M NaCl, and 0.005%, HBS. SPR capture experiments were performed by amine coupling 4000RU of anti-his monoclonal antibody (PHAb, 150mM NaCl, 20mM Tris pH 8, pentahis, Qiagen) on CM-5 research grade surfaces. C-terminal his-6 constructs sIL-22R1, sIL-22R1DDQ, IL-22BPI2, or IL-22BPI3, expressed as described by Logsdon et al. (Logsdon et al., 2004) were injected over the PHAb surface until a total of 120RU had been captured. Ten different concentrations of IL-22, including a buffer control, were injected over the sIL-22R1, IL-22BPI2, or IL-22BPI3 surfaces in random order at 50uL/min for 2 minutes and the dissociation phase was monitored for 7.5 minutes. Following each IL-22 injection, the PHab surface was cleaned with 0.1M glycine, 0.5M NaCl, pH 4.0 followed by fresh receptor and/or binding protein. Reproducible capture was monitored by pre- and post capture report points, which varied by less than 5 RU. The response curves were globally fit using BIAevaluation version 3.2, which gave equivalent parameters to data analysis using CLAMP version 3.1(Myszka and Morton, 1998). IL-22BPI2 off-rates were measured as previously described by Logsdon et al. (Logsdon et al., 2004) except, the chip surfaces were saturated by injecting 1µM IL-22BPI2 (2 min contact time) at 50uL / min and dissociation was followed using a data collection rate of 1 point per 10 seconds for 22 hours.
Supplementary Material
01
Acknowledgements
Use of the Advanced Photon Source (SERCAT ID-22) was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. W-31-109-Eng-38. This work was funded by grant AI047300 from the NIH.
Footnotes
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Accession Numbers:
Coordinates and structure factors have been deposited in the Protein Data bank with identification code 3DGC.
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