Structure and mechanism of receptor sharing by the IL-10R2 common chain

Abstract

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 ectodomain at 2.14 A 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.

Figure 1. Structure of the sIL-10R2 chain

(A) Ribbon diagram of the sIL-10R2 chain colored by secondary structure with binding loops labeled. Box shows the location of Figure F. (B) Superposition of sIL-10R2 (colored as in A) with sIL-10R1 (green) and sIL-22R1 (purple). Box shows the location of Figure D. (C) Location of aromatic residues on sIL-10R2 on sIL-10R2. (D) Comparison of the high affinity site 1 interaction between sIL-10R1 Y43^sIL-10R1 and the AB loop of IL-10 (green) with the sIL-10R2 L2 loop and Y59^sIL-10R2 (yellow). (E) Interaction network for K47^sIL-10R2. Replacement of K47^sIL-10R2 with a glutamic acid is associated with persistent HBV infection (Frodsham et al., 2006).

Figure 2. Unique clefts identified in sIL-10R2

The structures of sIL-10R2, sIL-10R1, and sIL-22R1 are shown as molecular surfaces. Images in the top row are orientated as found in Figure 1A. The surfaces are colored by atom type with oxygens red, nitrogens blue, and sulfur orange. Carbons are colored yellow, green, and purple in sIL-10R2, sIL-10R1, and sIL-22R1, respectively. Numbers on the surfaces correspond to loop positions.

Figure 3. Binding analysis of sIL-10R2 alanine mutants by SPR

(A) Relative binding of sIL-10R2 alanine mutants to IL-22/IL-22R1 (red), cmvIL-10/IL-10R1 (cyan), and hIL-10/IL-10R1 (black) binary complexes. sIL-10R2 mutant binding is presented in normalized response units (RU) relative to wild-type sIL-10R2 binding at concentrations of 150µM. Results are expressed as the mean of multiple measurements ±standard deviation. Residues tested for binding to IL-22/sIL-22R1 (B) and cmvIL-10/IL-10R1 (C) are mapped onto sIL-10R2 surfaces and colored according to the y axis in Figure A (See also Figures S3-S5).

Figure 4. IL-22 Ternary complex

(A) Overall structure of the IL-22 ternary complex. (B) Site 2 interactions between helix D and the L2/L3 cleft. (C) Site 2 interactions between helix A and L3/L5 cleft. (D) Site 3 contacts (E) Anti-Phospho-STAT3 western blot from lysates prepared from HepG2 cells stimulated with IL-22 or IL-22 mutants (See Also Figure S6 and Tables S1-S4).

Figure 5. cmvIL-10 Ternary complex

(A) Overall structure of the cmvIL-10 ternary complex. (B) Site 2 interactions between helix D and the L2/L3 cleft. (C) Site 2 interactions between helix A and the L3/L5 cleft. (D) Site 3 contacts. (E) Dimeric cmvIL-10 signaling complex. (See also Figures S6 and Tables S1-S4).

Figure 6. Common binding epitope identified between the promiscuous shared cytokine receptors

(A) Superposition of sIL-10R2 (yellow), gp130 (magenta), and γc chain (green). The inset shows the structural similarity of L3 loop residues Y82^sIL-10R2, F169^gp130 (F191^gp130 in uniprot database P40189), and Y103^γc. EPOR and the growth hormone receptor (GHR) also have aromatic L3 residues (F93^EPOR and W104^GHR) which have diverged from the structural alignment shown in Figure 6A (See Figure S7). (B) The position of Y82^sIL-10R2 and F169^gp130 are conserved in their respective ternary complexes. Ribbon diagram of the IL-22TC model (Fig 4) with IL-22/sIL-22R1 colored cyan and sIL-10R2 colored yellow. IL-6 from the IL-6/IL-6R/gp130 complex (pdbid 1P9M, (Boulanger et al., 2003)) was superimposed onto IL-22 from the IL-22TC model (Fig 4) and the position of gp130 (green) is shown. The inset shows IL-22 residues Y51^IL-22, and R55^IL-22 along with Y82^sIL-10R2 (yellow) and F169^gp130 (magenta).