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.