Mark R Walter

Department of Microbiology and Center for Macromolecular Crystallography, University of Alabama at Birmingham, Birmingham, Alabama

Interferon-y (IFN-y) is a pleotropic cytokine that induces antiviral, antiproliferative, and immunomodulatory effects on numerous target cells [1]. These diverse biological activities are initiated by IFN-y-mediated aggregation of at least two different cell surface receptors: IFN-yR1 and IFN-yR2. X-ray crystallographic studies of IFN-y and its receptors have been undertaken to delineate the molecular architecture of the receptor complexes and to understand the detailed recognition mechanisms that are ultimately responsible for IFN-y biological responses. Here, these structures are summarized in the context of current biochemical and bioactivity data.

Natural forms of human IFN-y are comprised of two 143-amino-acid peptide chains that are posttranslationally modified to contain an N-terminal pyroglutamic acid residue, N-linked glycosylation at two positions, and a heterogeneous C terminus containing the positively charged sequence KTGKRKR (residues 125-131). The crystal structure of human IFN-y has revealed the tight association of two peptide chains (comprised of six a-helices, labeled A to F, from the N to C terminus) into a remarkable intertwined helix topology to form a symmetric dimer (Fig. 1) [2]. As a result, the two-fold related domains of IFN-y are formed from the first four helices of one chain (A-D) and the last two helices (E' and F') from the other. Despite almost no sequence identity, the identical intertwined topology is also observed in the crystal structure of IL-10 [3]. The a-helices that form each domain are 9 to 21 residues long and are essentially linear (with the exception of helix F, which displays an « 50° bend). The helices are connected by short loops of 3 to 5 residues, except for the 13-residue AB loop that encircles helix F. C-terminal residues 124 to 143 extend away from the core of the molecule and are presumed to be flexible.

IFN-yR1 and IFN-yR2 are both type I membrane proteins that contain extracellular and cytoplasmic domains connected by a hydrophobic membrane spanning helix. The extracellular domain of IFN-yR1 binds IFN-y with high affinity («1 nM), while IFN-yR2 exhibits essentially no affinity for IFN-y. Coexpression of IFN-yR1 and IFN-yR2 on cells results in a fourfold increase in affinity for IFN-y compared to cells expressing IFN-yR1 alone, suggesting that the IFN-yR2 binding site is formed from residues on IFN-y and IFN-yR1 as presented in the IFN-y/IFN-yR1 complex. IFN-y-induced formation of the biologically active complex of IFN-y, IFN-yR1, and IFN-yR2 activates the Janus kinases (JAK1 and JAK2) that are associated with the cytoplasmic domains. JAK-dependent phosphorylation of the intracellular domain of IFN-yR1 results in the recruitment of the nuclear transcription factor STAT1 and subsequent expression of IFN-y-inducible genes [1].

At this time, structural information is only available for the extracellular domain of IFN-yR1 (sIFN-yR1) as it exists in complex with IFN-y (Fig. 1) [4-6]. Based on this work, sIFN-yR1 is comprised of two fibronectin type III domains (FnIII). The FnIII modules consist of a sandwich of two antiparallel P-sheets made up of seven P-strands: A, B, E, G, F, C, and C'. The N-terminal domain (D1) and the membrane-proximal or C-terminal domain (D2) are oriented at «120° to one another. The IFN-y binding site is located at the crevice between D1 and D2. Receptor residues that contact IFN-y are located on five different segments (labeled L2 to L6) that correspond to CC' and EF loops of D1, the domain linker, and BC and FG loops in D2.

In agreement with solution and cell-surface binding studies, the structure of the IFN-y/IFN-yR1 complex revealed the

Figure 1 Ribbon diagram of the IFN-y/IFN-yR1 receptor complex. The putative position of the cell membrane is at the bottom of the figure. The N and C termini of each molecule are labeled, as well as the D1 and D2 domains of the receptor.

Figure 2 The IFN-y/IFN-yR1 interface. IFN-yR1 is shown as a molecular surface while IFN-y segments (residues 1-34 and 108-138) that bind IFN-yR1 are represented by a yellow ribbon. Several IFN-yR1 residues that participating in important interactions in the interface are labeled by arrows. The sidechains of IFN-y residues Val-5, Glu-9, Arg-12, Ser-20, Asp-24, His-111, Glu-112, and Gln-115 are shown. The acidic patch located on IFN-yR1 is labeled, and the predicted interaction with a modeled C terminus of IFN-y is shown.

Figure 2 The IFN-y/IFN-yR1 interface. IFN-yR1 is shown as a molecular surface while IFN-y segments (residues 1-34 and 108-138) that bind IFN-yR1 are represented by a yellow ribbon. Several IFN-yR1 residues that participating in important interactions in the interface are labeled by arrows. The sidechains of IFN-y residues Val-5, Glu-9, Arg-12, Ser-20, Asp-24, His-111, Glu-112, and Gln-115 are shown. The acidic patch located on IFN-yR1 is labeled, and the predicted interaction with a modeled C terminus of IFN-y is shown.

symmetric binding of two IFN-yR1s to one IFN-y dimer (Fig. 1). The two receptors in the complex do not interact with one another and are separated by about 100 A at the putative position of the cell membrane. The large distance between the IFN-yR1s is consistent with the 1:2 complex being an intermediate that is dependent on IFN-yR2 binding and JAK2 recruitment to initiate the phosphorylation cascade. The two-fold symmetry of the IFN-y/IFN-yR1 complex suggests that it contains two binding sites for IFN-yR2.

The IFN-yR1 binding site is comprised of IFN-y residues 1 to 34 from one chain and residues 108 to 123 on the

Figure 3 Crystal structure of the 2:4 IL-10/IL-10R1 complex [7]. The view is looking down the two-fold axis of IL-10. The 1:2 IL-10/IL-10R1 complex represented by molecular surfaces is very similar to the IFN-y/IFN-yR1 complex shown in Fig. 1. The predicted positions of the IL-10R2s are shown as ribbons. The D1 and D2 domains for each receptor are labeled. This structure provides a possible model for the biologically active IFN-y/IFN-yR1/IFN-yR2 complex.

Figure 3 Crystal structure of the 2:4 IL-10/IL-10R1 complex [7]. The view is looking down the two-fold axis of IL-10. The 1:2 IL-10/IL-10R1 complex represented by molecular surfaces is very similar to the IFN-y/IFN-yR1 complex shown in Fig. 1. The predicted positions of the IL-10R2s are shown as ribbons. The D1 and D2 domains for each receptor are labeled. This structure provides a possible model for the biologically active IFN-y/IFN-yR1/IFN-yR2 complex.

other (Fig. 2). These residues form a continuous binding site that includes helix A, the AB loop, and helix B from one chain and helix F from the other. More recently, a 2-A structure of a mutant IFN-y/IFN-yR1 complex has revealed additional details of the binding site, including the participation of five ordered waters in the interface and the reassignment of IFN-yR1 Val-206 to an unfavorable phi-psi value to optimize its interactions with IFN-y [6]. In all free and bound structures reported to date, the C terminus of IFN-y that is important for binding and biological activity has not been observed; however, an acidic patch was identified on the receptor that may provide a "Velcro-like" interaction site for the basic C terminus of IFN-y [4].

Limited sequence identity confirms that IFN-yR2 is structurally similar to IFN-yR1; however, the inter-domain angle, receptor binding loops, and binding site cannot be accurately predicted. Currently, the most intriguing model for IFN-yR2 binding has been proposed from the analysis of the crystal structure of the IL-10/IL-10R1 complex [7]. In solution and the crystals, IL-10 and soluble IL-10R1 form a complex consisting of 2 IL-10 dimers and 4 IL-10R1s (Fig. 3). Structure and sequence comparisons of IL-10 and IFN-y receptors suggest that high-affinity (IL-10R1 and IFN-yR1) and low-affinity (IL-10R2 and IFN-yR2) receptors may share a common binding site on their respective cytokines. The 2:4 IL-10 receptor complex suggests a model for how the low-affinity IFN-yR2 may simultaneously interact with IFN-y and IFN-yRL This structural model is supported by limited homolog scanning on IFN-yR1 and radiation inactivation data showing that IFN-y biological activity requires two IFN-y dimers [1]. Confirmation of this model will require the structure determination of the IFN-y/IFN-yR1/IFN-yR2 complex.

References

1. Bach, E. A., Aguet, M., and Schreiber, R. D. (1997). The IFN-gamma receptor: a paradigm for cytokine receptor signaling. Annu. Rev. Immunol. 15, 563-591.

2. Ealick, S. E., Cook, W. J., Vijay-Kumar, S., Carson, M., Nagabhushan, T. L., Trotta, P. P., Bugg, C. E. (1991). Three-dimensional structure of recombinant human interferon-gamma. Science 252, 698-702.

3. Walter, M. R. (1997). Structural biology of cytokines, their receptors, and signaling complexes, in Blalock, J. E., Ed., Chemical ImmunologyNeuroimmunoendrocrinology, pp. 76-98. Karger, Basel.

4. Walter, M. R., Windsor, W. T., Nagabhushan, T. L., Lundell, D. J., Lunn, C. A., Zauodny, P. J., and Narula, S. K. (1995). Crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor. Nature 376, 230-235.

5. Thiel, D. J., le Du, M. H., Walter, R. L., D'Arcy, A., Chene, C., Fountoulakis, M., Garotta, G., Winkler, F. K., and Ealick, S. E. (2000). Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex. Struct. Fold Des. 8, 927-936.

6. Randal, M. and Kossiakoff, A. A. (2001). The structure and activity of a monomeric interferon-gamma: alpha-chain receptor signaling complex. Structure 9, 155-163.

7. Josephson, K., Logsdon, N. J., and Walter, M. R. (2001). Crystal structure of the IL-10/IL-10R1 complex reveals a shared receptor binding site. Immunity 14, 35-46.

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