Human IL-10 (hIL-10) modulates critical immune and inflammatory responses by way of interactions with its high-(IL-10R1) and low-affinity (IL-10R2) cell surface receptors. Human cytomegalovirus exploits the IL-10 signaling pathway by expressing a functional viral IL-10 homolog (cmvIL-10), which shares only 27% sequence identity with hIL-10 yet signals through IL-10R1 and IL-10R2. To define the molecular basis of this virus-host interaction, we determined the 2.7-Å crystal structure of cmvIL-10 bound to the extracellular fragment of IL-10R1 (sIL-10R1). The structure reveals cmvIL-10 forms a disulfide-linked homodimer that binds two sIL-10R1 molecules. Although cmvIL-10 and hIL-10 share similar intertwined topologies and sIL-10R1 binding sites, their respective interdomain angles differ by ϳ40°. This difference results in a striking re-organization of the IL-10R1s in the putative cell surface complex. Solution binding studies show cmvIL-10 and hIL-10 share essentially identical affinities for sIL-10R1 whereas the EpsteinBarr virus IL-10 homolog (ebvIL-10), whose structure is highly similar to hIL-10, exhibits a ϳ20-fold reduction in sIL-10R1 affinity. Our results suggest cmvIL-10 and ebvIL-10 have evolved different molecular mechanisms to engage the IL-10 receptors that ultimately enhance the respective ability of their virus to escape immune detection.

Summary 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.

Interleukin-22 (IL-22) is a cellular homolog of IL-10 that stimulates the production of acute-phase reactants. IL-22 and IL-10 require different ligand-specific receptor chains (IL-22R and IL-10R1) but share a second receptor chain (IL-10R2) to initiate cellular responses. The quaternary structures and the ability of IL-22 and IL-10 to engage soluble (s) IL-10R1, IL-22R, IL-10R2 receptor chains were analyzed using size exclusion chromatography and surface plasmon resonance techniques. In contrast to IL-10, which is a homodimer, IL-22 is a monomer in solution that forms a 1:1 interaction with sIL-22R. Kinetic binding data reveal sIL-22R and sIL-10R1 exhibit specific nanomolar binding constants for IL-22 (k(on)/k(off) = 14.9 nM) and a monomeric isomer of IL-10 (IL-10M1) (k(on)/k(off) = 0.7 nM), respectively. In contrast, IL-10R2 exhibits essentially no affinity for IL-22 (K(eq) approximately 1 mM) or IL-10M1 (K(eq) approximately 2 mM) alone but displays a substantial increase in affinity for the IL-10/sIL-10R1 (K(eq) approximately 350 microM) and IL-22/sIL-22R (K(eq) approximately 45 microM) complexes. Three-dimensional models of IL-22 and IL-10 receptor complexes suggest two receptor residues (Gly-44 and Arg-96) are largely responsible for the marked differences in ligand affinity observed for sIL-10R1 and sIL-22R vs. sIL-10R2.

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Summary IL-10R2 is a shared cell surface receptor required for the activation of five class 2 cytokines (IL-10, IL-22, IL-26, IL-28, and IL-29) that play critical roles in host defense. To define the molecular mechanisms that regulate its promiscuous binding, we have determined the crystal structure of the IL-10R2 ecto-domain at 2.14Ǻ resolution. IL-10R2 residues required for binding were identified by alanine scanning and used to derive computational models of IL-10/IL-10R1/IL-10R2 and IL-22/IL-22R1/IL-10R2 ternary complexes. The models reveal a conserved binding epitope that is surrounded by two clefts that accommodate the structural and chemical diversity of the cytokines. These results provide a structural framework for interpreting IL-10R2 single nucleotide polymorphisms associated with human disease.

Human IL-10 (hIL-10) is a cytokine that modulates diverse immune responses. The Epstein-Barr virus (EBV) genome contains an IL-10 homolog (vIL-10) that shares high sequence and structural similarity with hIL-10. Although vIL-10 suppresses inflammatory responses like hIL-10, it cannot activate many other immunostimulatory functions performed by the cellular cytokine. These functional differences have been correlated with the approximately 1000-fold lower affinity of vIL-10, compared to hIL-10, for the IL-10R1 receptor chain. To define the structural basis for these observations, crystal structures of vIL-10 and a vIL-10 point mutant were determined bound to the soluble IL-10R1 receptor fragment (sIL-10R1) at 2.8 and 2.7 A resolution, respectively. The structures reveal that subtle changes in the conformation and dynamics of the vIL-10 AB and CD loops and an orientation change of vIL-10 on sIL-10R1 are the main factors responsible for vIL-10's reduced affinity for sIL-10R1 and its distinct biological profile.

Ectromelia virus (ECTV) encodes an IFN-␥-binding protein (IFN-␥BPectromelia virus ͉ immunomodulator ͉ interferon ͉ cytokine ͉ complex E ctromelia virus (ECTV) is an orthopoxvirus that causes mousepox, which closely resembles the genetic and disease characteristics of the human pathogen variola virus (VARV), the causative agent of smallpox (1). The genomes of all orthopoxviruses, including ECTV and VARV, encode proteins required for viral replication, as well as soluble cytokine-and chemokine-binding proteins, which disrupt the activation and recruitment of immune cells responsible for host antiviral responses (2, 3). The severity of smallpox and mousepox has been attributed to the effectiveness of these immunomodulatory proteins, which in many instances, exhibit significant homology to cellular receptors, suggesting they were captured and adapted to subvert host immune responses during poxvirus evolution.All orthopoxviruses express IFN-␥-binding proteins (IFN␥BPs) that efficiently block IFN-␥-mediated signaling cascades responsible for activating potent antiviral defense mechanisms. The importance of IFN-␥ in viral pathogenesis is demonstrated by studies in C57BL/6 mice, in which depletion of IFN-␥ by monoclonal antibody treatment, or disruption of the signaling pathway through genetic means, transforms benign ECTV infection into a lethal one (4, 5). In addition, the ectromelia virus IFN-␥BP (IFN-␥BP ECTV ) has been shown to be a critical virulence factor in BALB/c mice, in which ECTV infections are lethal but infections with an ECTV mutant lacking a functional IFN-␥BP ECTV are not (6).Orthopoxvirus IFN-␥BPs are Ϸ270-aa proteins that share Ͼ90% sequence identity with one another and Ϸ20% sequence identity with the extracellular region of the cellular IFN-␥R1 chains, often called the cytokine receptor homology region (CRHR). In contrast to cellular IFN-␥R1s, which exhibit species-specific binding to their cognate ligand, IFN-␥BPs exhibit relaxed IFN-␥-binding specificity (7, 8) (e.g., IFN-␥BP ECTV binds human, murine, rabbit, and bovine IFN-␥). This functional difference may have facilitated opportunistic viral infections in multiple hosts during the evolution of the virus.In contrast to the CRHR, the C-terminal Ϸ60 aa of the IFN-␥BPs share no identifiable sequence similarity with cellular proteins. Recent studies suggest that the C terminus mediates oligomerization of the IFN-␥-binding domains (9). However, there are conflicting reports about the quaternary structure of the molecules. For example, IFN-␥BPs encoded by VACV-WR (Western Reserve) and myxoma virus (M-T7) have been reported to form dimers and trimers, respectively (10, 11). In contrast to these reports, we have demonstrated recently that IFN-␥BPs from ECTV and VACV-B8R (Copenhagen strain) adopt larger oligomers in solution, likely tetramers, which are critical for antagonizing IFN-␥ activity (9).To address the basic mechanisms of IFN-␥ antagonism by orthopoxvirus IFN-␥BPs, we determined the crystal structure of IFN-␥BP ECTV bound to human IFN-␥. IFN-␥...

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