Structural basis for receptor sharing and activation by
interleukin-20 receptor-2 (IL-20R2) binding cytokines
Naomi J. Logsdona, Ashlesha Deshpandea,b, Bethany D. Harrisa, Kanagalaghatta R. Rajashankarc,
and Mark R. Waltera,b,1
aCenter for Biophysical Sciences and Engineering andbDepartment of Microbiology, University of Alabama, Birmingham, AL 35294; andcNortheastern
Collaborative Access Team, Department of Chemistry and Chemical Biology, Cornell University, Argonne, IL 60439
Edited by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, and approved June 7, 2012 (received for review October 24, 2011)
Interleukin 20 (IL-20) is a pleotropic IL-10 family cytokine that
IL-20 signaling is implicated in several human pathologies including
psoriasis, rheumatoid arthritis, atherosclerosis, and osteoporosis.
IL-20, and related cytokines IL-19 and IL-24, designated IL-20 sub-
20R1/IL-20R2 (type I) receptor heterodimer, whereas IL-20 and IL-24
also signal through the IL-22R1/IL-20R2 (type II) receptor complex.
The crystal structure of the IL-20/IL-20R1/IL-20R2 complex reveals
how type I and II complexes discriminate cognate from noncognate
ligands. The structure also defines how the receptor–cytokine inter-
complex shared by three different ligands. Our results provide
unique insights into the complexity of IL-20SFC signaling that may
be critical in the design of mechanistic-based inhibitors of IL-20SFC–
mediated inflammatory disease.
1q32 in a cluster with IL-10, IL-19, and IL-24 (MDA7) (1). On
the basis of genomic location, amino acid sequence identity (32–
40%), and use of a common receptor chain for signaling (IL-
20R2), IL-19, IL-20, and IL-24 have been designated as IL-20
subfamily cytokines (SFCs) (2, 3). The IL-20 SFCs, IL-10, IL-22,
and IL-26 form the IL-10 cytokine family, which together with
the interferons (type I, IFN-α/β; type II, IFN-γ; and type III,
IFN-λs or IL-28/IL-29) form the class 2 cytokine family (1, 4, 5).
heterodimer composed of IL-20R1 and IL-20R2 chains (type I
complex) (Fig. 1) (1, 6, 7). IL-20 and IL-24 also signal through an
IL-22R1/IL-20R2 heterodimer (type II complex), whereas IL-19
only signals through the type I complex (6, 7). IL-20R1 and IL-
22R1 also pair with the IL-10R2 chain to form receptor hetero-
dimers that induce cell signaling upon IL-26 (IL-20R1/IL-10R2)
promiscuous pairing of the R2 chains, IL-20R2 cannot substitute
for IL-10R2 in IL-22 signaling (6). Furthermore, IL-19, IL-20, and
IL-24 appear to have largely nonredundant biological activities
in vivo, suggesting they may engage type I and type II receptor
heterodimers differently (6, 7, 11). However, a mechanistic basis
for such differences has not been determined.
IL-20 has been implicated in the pathophysiology of psoriasis
(1). Transgenic (Tg) mice overexpressing IL-20 exhibit a pheno-
type similar to human psoriatic skin, and IL-20 neutralizing
antibodies resolve psoriasis in a human xenograft transplation
model (1, 12). Increased levels of IL-19 and IL-24 are also ob-
served in skin samples from psoriasis patients, but the significance
and/or biological function of IL-19 and IL-24 is less clear (3).
IL-24 Tg mice exhibit epidermal hyperplasia and proliferation,
suggesting IL-24 activity in vivo is similar to IL-20 (13). However,
IL-19 Tg mice were reported to exhibit a normal skin phenotype,
which is consistent with IL-19’s unique receptor specificity. IL-20
is also implicated in rheumatoid arthritis (RA) (14) and athero-
sclerosis (15). IL-20 also exhibits arteriogenic/angiogenic prop-
erties and may be important for treating ischemic disease (16).
nterleukin 20 (IL-20) is an α-helical cytokine discovered by
EST database mining and mapped to human chromosome
Most recently, IL-20 was found to induce osteoclastogenesis, by
up-regulating the receptor activator of NFκB (RANK)–RANK
ligand signaling proteins and may be a therapeutic target for os-
Expression of the IL-20R1/IL-20R2 and IL-22R1/IL-20R2
heterodimers, which are required for IL-20 bioactivity, have been
observed only on cells of epithelial origin including skin, lung, and
testis (1, 18, 19). These data suggest a major role for IL-20 SFCs
in mediating cross-talk between infiltrating immune cells (T cells,
macrophages, and dendritic cells) that express the IL20SFCs, and
the skin. However, in some cases the pleotropic activities of IL-20
are at odds with the cellular expression of the IL-20 receptors. In
particular, IL-20 and IL-19 induce naïve T cells toward a TH2
secretory phenotype, characterized by increased IL-4, IL-13, and
reduced IFNγ production (20, 21). However, only IL-20R2 but
not IL-20R1 or IL-22R1 have been detected in immune cells (1,
18, 19). In addition, IL-20R2 knockout mice exhibit disrupted
CD4+and CD8+T-cell function (22), which implicates the IL-20
SFCs in T-cell signaling, despite the absence of the IL-20R1 and
IL-22R1 receptor chains on these cells (18). On the basis of these
data, it has been hypothesized that another receptor chain must
pair with IL-20R2 to induce IL-20SFC signaling on immune cells.
IL-20 has emerged as a highly pleotropic cytokine involved in
essential cellular processes and pathology. Despite an improved
understanding of IL-20 biology, the molecular mechanisms that
allow IL-20, IL-19, and IL-24 to discriminate and activate type I
(IL-20R1/IL-20R2) and type II (IL-22R1/IL-20R2) receptor
complexes are currently unknown. To address this question, we
have determined the crystal structure of the IL-20/IL-20R1/IL-
20R2 complex to determine how signaling complexity can be
obtained for structurally similar cytokines.
Overall Architecture of the IL-20/IL-20R1/IL-20R2 Signaling Complex.
The structure of IL-20/IL-20R1/IL-20R2 (Fig. 2A) was solved at
2.8´Å by single wavelength anomalous diffraction phasing and
molecular replacement methods (Table S1). IL-20 adopts an
α-helical fold, which is highly conserved with IL-19 (rmsd 0.79´Å).
Despite structurally similar helical cores, the N terminus of IL-20
adopts a novel β-hairpin structure, rather than the 310helix/coil
structure observed in IL-19 (Fig. 2B). Threading the IL-24 amino
acid sequence onto the structure of IL-20 suggests IL-20 and
IL-24 adopt similar N-terminal β-hairpin structures, which
positions Cys-59IL-24and Cys-106IL-24in close proximity (Cα–Cα
distance = 6.6´Å) for disulfide bond formation (SI Materials and
Author contributions: N.J.L., A.D., B.D.H., K.R.R., and M.R.W. designed research; N.J.L.,
A.D., B.D.H., K.R.R., and M.R.W. performed research; N.J.L., A.D., K.R.R., and M.R.W.
analyzed data; and M.R.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
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Methods, Fig. S1). Thus, the predicted structural similarity of
IL-20 and IL-24 is consistent with the identical receptor binding
profiles of each cytokine (Fig. 1).
The extracellular fragments of IL-20R1 and IL-20R2 each
consist of tandem β-sandwich domains (D1 and D2) that as-
semble around IL-20 to form a V-shaped complex, when viewed
down the α-helical bundle axis of IL-20 (Fig. 2A). X-ray analysis
of IL-20R1 and IL-20R2 completes the structural descriptions of
all five IL-10 family receptors (23–26) (Fig. 1). Structural com-
parisons of the receptors reveal the three R1 chains are highly
similar to one another, whereas IL-10R2 and IL-20R2 exhibit
structurally divergent cytokine binding loops and interdomain
angles (Fig. 2C and SI Materials and Methodsand Fig. S2).
IL-20/IL-20R1/IL-20R2 ternary complex formation is medi-
ated by three protein interfaces, IL-20/IL-20R1 (site 1), IL-20/
IL-20R2 (site 2), and IL-20R1/IL-20R2 (site 3), that bury a total
of 4,236´Å2of accessible surface area (Fig. 2A). The IL-20/IL-
20R1 site 1 interface (1,576´Å2) consists of two contact surfaces,
site 1a and site 1b (Fig. 3A). Site 1a is formed by IL-20R1 L2–L4
loops that contact a small cavity on IL-20 located at the in-
tersection of helix F and AB loop. Site 1b contacts occur between
of the total buried surface area and eight of nine hydrogen bond
(H-bond)/salt bridge interactions identified in the IL-20/IL-20R1
small and made up almost entirely of IL-20R1 L6 residues Gly-
224IL-20R1, Pro-225IL-20R1, and Pro-226IL-20R1, which form Van der
Waal contacts with the aliphatic portion of Arg-43IL-20and a single
hydrogen with helix A residue Gln-40IL-20.
The site 2 IL-20/IL-20R2 interface (1,624´Å2) is centered on
IL-20 helix D, which is surrounded by IL-20R2 L2 and L3 loops
(Fig. 4). The IL-20R2 L2 loop also makes significant contacts
with helix C, via Tyr-74IL-20R2, whereas residues on L4 and L5
loops form hydrogen bonds with helix A and the IL-20 N ter-
minus. As observed in site 1a, the IL-20R2 L2 loop forms the
majority of contacts in site 2 by contributing 57% of the buried
surface area, and 9 of 13 hydrogen bonds in the IL-20/IL-20R2
interface (Table S3). Six of these hydrogen bonds are formed
with helix D residues Ser-110IL-20, Ser-111IL-20, and Asn-114IL-20,
which are conserved in the sequences of IL-19 and IL-24 (Fig. 4B
and SI Materials and Methods and Fig. S1). Thus, IL-20R2, and
the IL-20SFCs share a conserved binding epitope to facilitate
promiscuous IL-20R2 ligand engagement (Fig. 1).
Essentially all contacts between IL-20 and the receptors
are mediated by the D1 domains, whereas IL-20R1 and IL-20R2
D2 domains contact one another to form the base of the V-
shaped complex (site 3, Figs. 2A and 5A). The site 3 interface
(1,036´Å2) is formed from IL-20R1 D2 residues on β-strand C′,
the CC′ loop, and the EF loop, which contact IL-20R2 residues
on the AB loop, β-strand E, and the EF loop. The interface is
quite extensive, including 6 hydrogen bonds (Table S4) con-
tributed predominantly from the AB loop of IL-20R2 and the
EF loop of IL-20R1.
IL20/IL-20R1/IL-20R2 Dimer Complex. Two 1:1:1 IL-20/IL-20R1/IL-
20R2complexesforma dimer inthe asymmetricunitofthecrystals
(Fig. 2D). The IL-20 dimer interface is very small (∼200´Å2), con-
sistent with IL-20 being a monomer in solution. The dimer is sta-
bilized by contacts formed between IL-20R2 chains that bridge the
2A and 4 and Table S5), and a second contact between IL-20R2
D2 domain andIL-20 helix D(residues 121–130, Fig. S3). The IL-
20/IL-20R2 dimer interface is extensive, burying 1,982´Å2of ac-
cessible surface area. Two IL-20R1 chains are also in the com-
plex, but they do not contribute residues to the dimer interface.
Although the formation of cell surface IL-20R2 dimers might
provide an explanation for IL-20SFC signaling on immune cells
that apparently lack IL-20R1 (27), we have been unable to detect
an IL-19/IL-20R2 dimeric complex in solution or identify signif-
icant differences in IL-20SFC binding affinity between mono-
meric and dimeric IL-20R2 (SI Materials and Methods, Fig. S4
and Table S6).
Mechanisms Regulating IL-20R1 Chain Affinity. Surface plasmon
resonance (SPR) experiments were performed to quantify IL-
20R1 binding to IL-19 and IL-20 (Fig. 6). We could not detect
binding between IL-19 and IL-20R1 at IL-19 concentrations up
to 10 μM. However, a binding constant of ∼9 μM was determined
for the IL-20/IL-20R1 interaction (Fig. 6B). In contrast to very
weak IL-20R1 interactions, IL-10R1 and IL-22R1 chains exhibit
∼10,000-fold tighter binding affinity for IL-10 (kDa = 0.5 nM)
and IL-22 (kDa = 1.2 nM) (24, 28) (Fig. 1). These extremely
different binding affinities occur despite structurally similar
IL-20/IL-20R1, IL-22/IL-22R1, and IL-10/IL-10R1 binary com-
plexes (Fig. 3B). Comparison of each interface reveals identical
“YG” interaction motifs consisting of receptor YG residues (L2
loop Tyr-76sIL-20R1and Gly-77sIL-20R1in IL-20R1, Fig. 3A) that
are inserted into a conserved cleft formed by the AB loop and
distinguishes IL-20R2 signaling complexes from IL-10R2 complexes, which use
identical R1 chains.
Schematic diagram of IL-10 family receptor complexes. The box
IL-20 ternary signaling complex. (B) Ribbon diagram of IL-19 and IL-20, with
unique N-/C-terminal regions colored yellow and magenta, respectively. (C)
Superposition of the R1 chains, IL-10R1 (cyan), IL-20R1 (red), and IL-22R1
(magenta), on the Left and R2 chains, IL-10R2 (yellow) and IL-20R2 (green)
on the Right. (D) The IL-20 dimeric complex observed in the asymmetric unit
of the crystals (SI Materials and Methods and Fig. S3).
IL-20/IL-20R1/IL-20R2 ternary complex. (A) Ribbon diagram of the
Logsdon et al. PNAS
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helix F of the ligands (Fig. 3 C and D) (23, 24, 26). Buried sur-
face area does not predict the observed affinities of the different
complexes (Table S7). However, complex affinities correlate with
unique contacts made by four receptor residues (D1 residues
Phe-74 (L2 loop), Glu-105 (L3 loop), Tyr-109, and Arg-128 (L4
loop) in IL-20R1, Fig. 3 C and D; for clarity, IL-20R1 numbering
will be used throughout the text), located adjacent to the YG
interaction motif (Fig. 3 C and D).
In the high-affinity IL-10/IL-10R1 complex, Arg-109IL-10R1
and Arg-128IL-10R1, adjacent to the YG motif, form a network of
four hydrogen bonds with IL-10 residues Gln-38IL-10and Asp-
166IL-10(Fig. 3C). However, in IL-20/20R1 and IL-22/IL-22R1
interfaces, Arg109IL-10R1is replaced with Tyr-109IL-20R1, IL-22R1,
which no longer contacts IL-20 or IL-22 directly, but repositions
Arg-128IL-20R1, IL-22R1for interactions with Asp-57IL-20, IL-22,
despite the fact Asp-166 is conserved in IL-20 and IL-22 (Fig.
3D). Thus, this network of four hydrogen bonds in the IL-10/IL-
10R1 interface is reduced to one in IL-20/IL-20R1 and IL-22/IL-
To compensate for the loss of an “IL-10 like” bonding net-
work, the IL-22/IL-22R1 interface forms a unique three hy-
drogen bond network on the opposite side of the YG motif (Fig.
3D). This interaction includes hydrogen bonds between Arg-
63IL-22and Glu-105IL-22R1and between IL-22R1 L2 loop resi-
due, Lys-74IL-22R1, and IL-22 AB loop residues Thr-60IL-22and
Asp-61IL-22. Mutation of Lys-74IL-22R1to alanine reduces IL22
functional activity by ∼100-fold, supporting a critical role for
this bonding network in IL-22/IL-22R1 interactions (26).
Consistent with its low affinity and in contrast to IL-10/IL-
10R1 and IL-22/IL-22R1, the IL-20/IL-20R1 complex does not
form additional site 1a hydrogen bonding networks (Fig. 3D).
Lys-74IL-22R1, the key residue in the IL-22/IL-22R1 interface, is
replaced by Phe-74IL-20R1and Thr-60IL-22is replaced with Ile-
60IL-20. Thus, the IL-20/IL-20R1 site 1a interface consists of the
YG motif and two additional hydrogen bonds (Arg-128IL-20R1/
Asp-57IL-20and Glu-105IL-20R1/Arg-63IL-20) that are conserved
with IL-22/IL-22R1. Additional disruption of these interactions is
predicted in the lower affinity IL-19/IL-20R1 interface where
and Thr63IL-19in IL-19.
Mechanisms Regulating IL-20R2 Binding to IL-19 and IL-20. Additional
SPR experiments reveal IL-19 binds tighter to IL-20R2 (kDa =
gram of the IL-20/IL-20R1 interface. (B) Superposition of IL-10/IL-10R1 (cyan),
IL-20/IL-20R1 (yellow), and IL-22/IL-22R1 (magenta) binary complexes. (C)
Comparison ofsite1abetween IL-10/IL-10R1 (cyan)andIL-20/IL-20R1 (yellow).
IL-20 and selected residues are colored magenta. Residues corresponding to
IL-10 are colored purple and shown on the IL-20 scaffold. Dashed lines in the
circle, colored cyan, are IL-10–specific hydrogen bonds. Black dashed lines
correspond to IL-20/IL-20R1 hydrogen bonds. (D) Comparison of site 1a be-
tween IL-22/IL-22R1 (tan) and IL-20/IL-20R1 (yellow). IL-20 is colored as in C.
IL-22 residues are green and shown on the IL-20 scaffold. Dashed lines in the
circle, colored tan, are IL-22–specific hydrogen bonds. Black dashed lines
correspond to hydrogen bonds conserved in IL-20 and IL-22 interfaces. (E and
F) Site 1b steric clashes observed for noncognate complexes (Fig. 1). IL-19 (E)
clashes with IL-22R1 L6 loop (green), but not IL-20R1 L6 (magenta). IL-22 (F)
clashes with the IL-20R1 L6 loop (magenta), but not IL-22R1 L6 (green).
Site 1 interface: structure, affinity, and specificity. (A) Ribbon dia-
(A) Ribbon diagram of the IL-20/IL-20R2 interface. (B) Detailed contacts in the
IL-20/IL-20R2 site 2 interface. (C) Conformational differences in helix C be-
tween IL-20 bound to IL-20R2 (magenta) and unbound IL-19 (blue). The
IL-20R2 L2 loop (green) is also shown. (D) Contacts between IL-19 (blue) and
IL-20R2 (green), not found in IL-20 (magenta)/IL-20R2, that modulate in-
creased IL-19/IL-20R2 affinity. (E) Specificity mechanism preventing non-
cognate IL-22 (yellow) from binding to IL-20R2 (green) is controlled by IL-22
helix D residue, Phe-105IL-22. Cognate IL-20 (magenta) is also shown (Fig. 1).
IL-20 site 2 interface: mechanisms controlling affinity and specificity.
| www.pnas.org/cgi/doi/10.1073/pnas.1117551109Logsdon et al.
105 nM) than IL-20 (kDa = 697 nM) (Fig. 6 C and D). Super-
position of unbound IL-19 onto IL-20 in the ternary complex,
reveals most interactions observed in the IL-20/IL-20R2
interface (Fig. 4B and Table S2) are conserved in the IL-19/
IL-20R2 complex. However, helix C adopts a different conforma-
tion (∼3´Å changes) in IL-19, relative to IL-20 bound to IL-20R2
(Fig. 4C). The conformation of helix C in IL-19 positions Asp-
92IL-19into hydrogen bonding distance (3.5´Å) with the OH of
Tyr-78IL-20R2, whereas the equivalent residue in IL-20 (Asp-
92IL-20) is 4.3´Å from Tyr-78IL-20R2(Fig. 4D). Replacement of
IL-20 Lys-120IL-20with Gln-120IL-19in IL-19 forms an addi-
tional hydrogen bond Tyr-78IL-20R2in the IL-19/IL-20R2. The
additional hydrogen bonds in the IL-19/IL-20R2 interface are con-
sistent with the higher affinity of this complex than IL-20/IL-20R2
(Fig. 6 C and D). The structural differences between IL-19 and
IL-20 demonstrate helix C and the CD loop are conformationally
dynamic, which may also influence IL-20R2 binding affinity.
Structural Mechanisms Regulating Cognate vs. Noncognate Ligand
Specificity. IL-20R1 and IL-22R1 form promiscuous interactions
with five different cytokines and two R2 chains to engage dif-
ferent signaling responses that protect the host from invading
pathogens (Fig. 1). These cognate ligand receptor complexes
have been “affinity tuned,” using mechanisms described above,
to optimize their signaling properties. However, IL-19 cannot
bind or signal through the IL-22R1 chain. Furthermore, IL-22
cannot bind or induce signaling through the IL-20R1 chain (Fig.
1). Superposition of IL-22 onto IL-20/IL-20R1 reveals the major
specificity determinant is site 1b, where the IL-20R1 L6 loop
forms steric clashes with IL-22 helix A residues Phe-57IL22and
Asn-54IL-22(Fig. 3F). Similar clashes are observed between IL-
22R1 L6 and the N terminus of IL-19 (Fig. 3E), which explains
why IL-19 cannot signal through the type II complex (Fig. 1).
Thus, although cognate site 1b contacts are not always extensive,
they are clearly critical in determining ligand–receptor specific-
ity. These results provide additional evidence for a two-point
(site 1a/site 1b) R1 chain specificity mechanism described by
Jones et al. (24).
Signaling specificity is also achieved through the IL-20R2
chain, which must engage IL-19, IL-20, and IL-24, but prevent
IL-22 signaling through the type II (IL-22R1/IL-20R2) complex
(Fig. 1) Because IL-22 can signal through the IL-22R1/IL-10R2
heterodimer, but not the IL-22R1/IL-20R2 complex (6), speci-
ficity must occur in the IL-22/IL-20R2 interface. To test this
hypothesis, IL-22 was positioned onto IL-20 in the IL-20 ternary
complex. This experiment positions IL-22 helix C residue, Phe-
105IL-22, at the center of the site 2 interface, where it forms ex-
tensive steric clashes with IL-20R2 residues Tyr-74IL-20R2and
Tyr-78IL-20R2(Fig. 4E). These steric disruptions would prevent
noncognate IL-22/IL-20R2 interactions and subsequent cellular
signaling by this complex.
Importance of the Site 3 Interface in Complex Formation and Signaling.
The low affinity of IL-20R1 and IL-20R2 binary complexes
emphasizes the importance of the site 3 interface (Figs. 2A and
5A) in forming signaling competent ternary complexes. To esti-
mate the stability of IL-20 (IL-20/IL-20R1/IL-20R2 and IL-20/IL-
22R1/IL-20R2) and IL-19 (IL-19/IL-20R1/IL-20R2) ternary
complexes (Fig. 1), IL-19 or IL-20 was coinjected with soluble IL-
20R1 over a biacore chip coupled with IL-20R2. The apparent
affinities obtained from the coinjection experiments (Fig. 6 E–H)
suggest the IL-20 type I complex (Fig. 1) is the most stable
(KDapp) of 319 nM, followed by the IL-20 type II complex
(KDapp = 1,363 nM), and then the IL-19/IL-20R1/IL-20R2
complex (KDapp = 3,121 nM). The same results, although with
different KDapp values, were obtained by evaluating ternary
complex stability by injecting soluble IL-20R2 and IL19, or IL-20,
over biacore chip surfaces of IL-20R1 or IL-22R1 (SI Materials
and Methods and Fig. S5).
To further test the importance of the site 3 interface, three IL-
20R1 site 3 residues that form site 3 contacts (Fig. 5A, Ser-
190IL20R1, Trp- 194IL-20R1, and Trp-207IL-20R1) were mutated to
alanine to create an IL-20R1 triple mutant, M3. Coinjection of
IL-19+M3 over an IL-20R2 biacore surface, generated the same
binding response as IL-19 alone (Fig. 5B). Coinjection of IL-20+
M3 exhibited an increased binding response, relative to IL-20
alone, although it was drastically reduced from the injection of
IL-20+IL-20R1. The increased binding observed for the IL-20+
M3 injection corresponds to IL-20/M3 binary complexes binding
to IL-20R2 with the same kinetics as the IL-20/IL-20R2 in-
teraction (e.g., without the site 3 interaction). This result con-
firms the higher affinity of the IL-20/IL-20R1 site 1 interface
(Fig. 6 A and B), relative to IL-19/IL-20R1, and the importance
of site 3 in forming stable ternary complexes essential for
The essential role of the site 3 interface in IL-20 ternary
complex formation, led us to examine how the IL-20R1/IL-20R2
D2/D2 interface (site 3) differed from other cytokine ternary
complexes. To ask this question, the D2 domains of growth
hormone receptor (GHR, site 1) (29) and IL-6R (30) were
superimposed onto IL-20R1 D2 and the orientations of GHR
(site 2) and GP130 D2 domains were evaluated (Fig. 5C). The
GHR/GHR D2 domains are essentially parallel to one another
and were assigned a D2–D2 crossing angle of 0°. Compared with
Site 3 interface with IL-20R1 colored green and IL-20R2 colored yellow. (B) Im-
portance of the site 3 interface in ternary complex formation. SPR sensograms
obtained by injecting cytokines (IL-19 or IL-20, at 250 nM) and/or receptors (IL-
20R1 or M3, at 425 nM), as labeled, over an IL-20R2 coupled biacore chip. In-
jection of IL-20+IL-20R1 (black line) results in a large response, consistent with
ternary complex formation. However, injection of IL-20+M3 (green line), an IL-
20R1 site 3 mutant with S190IL-20R1, W194IL-20R1, and W207IL-20R1(A) mutated to
alanine, drastically reduces binding due to disruption of ternary complex for-
mation. Similar results are observed for complex formation by IL-19. (C) Su-
perposition of the D2 domains of GHR site 1, IL-6R, and IL-20R1 (cyan). The
(yellow) are shown, along with a schematic of the differences in their ori-
entations.(D) Putative modelof the IL-22/IL-22R1/IL-10R2 complexbasedon the
IL-20 ternary complex structure (Fig. 2A, SI Materials and Methods and Fig. S5).
The D2 domains of the model are separated by 15´Å, suggesting IL-10R2 com-
plexes are distinct from IL-20R2 complexes.
IL-20 site 3 interface is essential and distinct from other complexes. (A)
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GHRs, the D2 domains of IL-20R1/IL-20R2 and IL-6R/GP130
D2 cross at angles of −40° and +25°, respectively (Fig. 5C).
These differences may be important as the D2 domains are lo-
cated adjacent to the membrane where they could selectively
influence intracellular signal transduction pathways and ulti-
mately cellular responses.
Not only is the IL-20/IL-20R1/IL-20R2 complex distinct from
the distantly related class-1 cytokine complexes, but it is also
distinct from IL-10R2-containing complexes (Fig. 1). For exam-
ple, superposition of the IL-22/IL-22R1 binary complex and IL-
10R2onto IL-20/IL-20R1/IL-20R2 results in a IL-22/IL-22R1/IL-
10R2 complex that does not form a site 3 interface (Fig. 5D). This
result is caused by the distinct interdomain angle of IL-20R2
compared with IL-10R2 (SI Materials and Methods and Fig. S2).
To determine whether IL-10R2 adopts a different interdomain
angle in solution, compared with the crystal structure (25), solu-
tion small angle X-ray scattering (SAXS) was performed on IL-
10R2.Theseexperiments confirmthat IL-10R2insolution adopts
the same D1/D2 interdomain angle observed in the crystal
structure (Fig. S6). These studies further underscore the unique
architectures of the IL-20R2 and IL-10R2 ternary complexes and
their distinct assembly properties.
The crystal structure of IL-20/IL-20R1/IL-20R2 depicts a com-
plete signaling complex of an IL-10 family cytokine, although two
binary complexes (IL-10/IL-10R1 and IL-22/IL-22R1) have been
determined (23, 24, 26). The surprising structural differences
betweenIL-19 andIL-20at theN- andC termini(SIMaterials and
Methods and Fig. S1) explains how structurally similar cytokines
discriminate, via IL-20R1 and IL-22R1, between the type I and
type II receptor heterodimers (Fig. 4 E and F). In contrast, IL-22,
which binds tightly to IL-22R1 (24), cannot signal through the
type II complex due to steric clashes between IL-22/IL-20R2 in
site 2, especially with Phe-105IL22(Fig. 5E). These results provide
a structural basis for IL-20SFC receptor specificity, which con-
tributes to their distinct in vivo biological properties.
In addition to discriminating between the type I and type II
complexes (Fig. 1), IL-19 and IL-20 modulate their biological ac-
tivities through distinct affinities for IL-20R1 and IL-20R2 chains
(Fig. 6). Our structural studies now provide a molecular basis for
how subtle structural rearrangements of the receptor interfaces,
combined with amino acid substitutions, alter ligand receptor
binding affinity. Prior experiments revealed IL-20R2 binds tighter
to IL-19 and IL-20 than IL-20R1 (7, 31). However, in contrast
to ∼1 nM IL-10/IL-10R1 and IL-22/IL-22R1 affinities (Table S7),
IL-19/IL-20R2 and IL-20/IL-20R2 interactions are at least ∼100-
fold weaker. Thus, in contrast to IL-10/IL-10R1 and IL-22/IL-
22R1 complexes (Fig. 1), the IL-20R2 chain does not dominate
ligand binding energetics, but must rely on cooperation between
IL-20R1 and IL-20R2 to assemble the signaling complex. This
property may allow the type-I receptor heterodimer (Fig. 1) to
selectively discriminate IL-19, IL-20, and IL-24 affinity differences
and induce distinct cellular responses. Using SPR (Fig. 6), the
stability ofthe IL-20type-I andtype-II complexeswerefound tobe
more stable than the IL-19 type-I complex (Figs. 1 and 6). The
molecular stabilities of the ternary complexes (IL-20 > IL-19) are
consistent with robust IL-20 signaling in keratinocytes and its pu-
tative role in psoriasis, properties not shared by IL-19 (1, 7).
The presence of an IL-20/IL-20R1/IL-20R2 dimer in the
crystals (Fig. 2D) provided a possible explanation for how IL-
20SFCs might signal on immune cells in the absence of IL-20R1
(20, 22, 27). However, we could not generate biochemical evi-
dence to confirm IL-19 or IL-20 can dimerize IL-20R2 in the
absence of IL-20R1 (SI Materials and Methods and Fig. S4). Our
studies suggest the main signaling unit of IL-19 and IL-20 are the
ternary complexes, which may further oligomerize on cells. How
the IL-20SFCs induce signals on immune cells, apparently in the
absence of IL-20R1, remains to be elucidated (20, 27).
Materials and Methods
Protein Expression and Purification of the Complex. IL-20 (residues 25–176,
uniprot Q9NYY1) was expressed with an N-terminal histidine tag in insect
cells. IL-20R1 (residues 29–245, uniprot Q9UHF4), and IL-20R2 (residues 30–231,
uniprot Q6UXL0) were expressed in insect cells with C-terminal histidine tags.
Two N-linked glycosylation attachment sites in IL-20R2, (Asn-40IL-20R2and
Asn-134IL-20R2) were removed by mutagenesis (Quick Change; Stratagene)
converting the asparagines to glutamines to yield IL-20R2QQ, used for crys-
tallization studies. IL-20R1 was modified by mutation of Lys-111IL-20R1and Lys-
113IL-20R1to arginines (IL-20R1RR) for crystallization. IL-20, and the receptors,
were purified by nickel affinity chromatography. The histidine tags of all
three proteins were removed by incubation with FactorXa protease. The
errors obtained for complexes described at the Top of each panel. Panels are labeled as injected soluble proteins, followed by a semicolon (:), then the protein
coupled to chip surface, IL-20R1 or IL-20R2. (A and B) Injection of IL-19 and IL-20 (maximum concentration, max conc. = 10 μM, twofold diluted to 0.625 μM)
over IL-20R1. (C and D) Injection of IL-19 (max conc. = 500 nM, twofold diluted to 3.91 nM) or IL-20 (max conc. = 2,000 nM, twofold diluted to 62.5 nM) over IL-
20R2. (E–H) IL-19 and IL-20 (fixed at 250 nM) were mixed with different concentrations of IL-20R1 or IL-22R1, (max conc. = 1,000 nM, twofold diluted to 7.81
nM) and injected over IL-20R2. To estimate R1 chain affinity/complex stability, the contribution of the ligand was removed by using the 250 nM IL-19 or IL-20
sensorgram as the blank subtraction. All sensorgrams were fit to 1:1 binding models. Kinetic parameters are listed in Table S6.
IL19/IL-20 receptor interactions and complex stability. SPR sensorgrams, data fit (black lines), equilibrium dissociation constant values, and residual
| www.pnas.org/cgi/doi/10.1073/pnas.1117551109 Logsdon et al.
individual proteins were incubated at approximately a 1:1:1 stoichiometric
ratio and purified by gel filtration chromatography. Fractions containing the
ternary complex were concentrated to 7 mg/mL for crystallization.
Crystallization, X-ray data collection and refinement, surface plasmon
resonance, and SAXS experiments are described in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Jean-Christophe Renauld for IL-20R2
cDNA, Sergei Kotenko for IL-20R1 cDNA, and Joshua LaBaer, Institute of
Proteomics, Harvard Medical School, for IL-20 cDNA. This work was funded
by National Institutes of Health Grants RO1-AI047300 and AI0473-S1 (to
M.R.W.) The Advanced Photon Source, Southeastern Collaborative Access
Team, beamline ID-22 is supported by contract no. W-31-109-Eng-38. Struc-
ture factors and coordinates have been deposited in the Protein Data Bank
(accession nos. rcsb070584 and 4DOH). Usage of the Biacore T-200 is made
possible by the University of Alabama at Birmingham Multidisciplinary Mo-
lecular Interaction Core. The Structurally Integrated BiologY for Life Sciences
beamline is supported by the Department of Energy via the Integrated Dif-
fraction Analysis Grant (DE-AC02-05CH11231).
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| vol. 109
| no. 31