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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 8 (April 15), 2000:
pp. 2491-2498
PLENARY PAPER
Structure of the activation domain of the GM-CSF/IL-3/IL-5
receptor common  -chain bound to an antagonist
Jamie Rossjohn,
William J. McKinstry,
Joanna M. Woodcock,
Barbara J. McClure,
Timothy R. Hercus,
Michael W. Parker,
Angel F. Lopez, and
Christopher J. Bagley
From the Ian Potter Foundation Protein Crystallography
Laboratory, St. Vincent's Institute of Medical Research, Fitzroy,
Victoria, Australia; the Cytokine Receptor Laboratory and the
Protein Laboratory, Hanson Centre for Cancer Research,
Institute of Medical and Veterinary Science, Adelaide, South Australia,
Australia; and the Royal Adelaide Hospital, Adelaide, South Australia,
Australia.
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Abstract |
Heterodimeric cytokine receptors generally consist of a major
cytokine-binding subunit and a signaling subunit. The latter can
transduce signals by more than 1 cytokine, as exemplified by the
granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the
signaling subunits in isolation are unable to bind cytokines, a fact
that has made it more difficult to obtain structural definition of
their ligand-binding sites. This report details the crystal structure
of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor  -chain
(c) signaling subunit in complex with the Fab fragment
of the antagonistic monoclonal antibody, BION-1. This is the first
single antagonist of all 3 known eosinophil-producing cytokines, and it
is therefore capable of regulating eosinophil-related diseases such as
asthma. The structure reveals a fibronectin type III domain, and the
antagonist-binding site involves major contributions from the loop
between the B and C strands and overlaps the cytokine-binding site.
Furthermore, tyrosine421 (Tyr421), a key
residue involved in receptor activation, lies in the neighboring loop
between the F and G strands, although it is not immediately adjacent to
the cytokine-binding residues in the B-C loop. Interestingly,
functional experiments using receptors mutated across these loops
demonstrate that they are cooperatively involved in full receptor
activation. The experiments, however, reveal subtle differences between
the B-C loop and Tyr421, which is suggestive of distinct
functional roles. The elucidation of the structure of the
ligand-binding domain of c also suggests how different
cytokines recognize a single receptor subunit, which may have
implications for homologous receptor systems.
(Blood. 2000;95:2491-2498)
© 2000 by The American Society of Hematology.
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Introduction |
Cytokine receptors exist largely as homodimers or
heterodimers.1-3 Homodimeric receptors, of which the human
growth hormone receptor (GH) is the prototype, bind to a single
cytokine that bridges 2 identical subunits and causes receptor
activation.2 In contrast, heterodimeric receptors comprise
2 or 3 subunits that subserve distinct and specialized functions: a
major ligand-binding subunit (the  subunit) and a signaling subunit
(the or  subunit). Importantly, a signaling subunit is able to
recognize several cytokines complexed to the appropriate -chain and
to transduce their signals. This is exemplified by the common -chain
(c) of the granulocyte-macrophage colony-stimulating
factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors; the common
IL-2 receptor -chain (shared by the IL-2, IL-4, IL-7, IL-9, and
IL-15 receptors); and gp130 (shared by the receptors for IL-6, IL-11,
Leukemia Inhibitory Factor (LIF), ciliary neurotrophic factor,
oncostatin M, and cardiotrophin).
The GM-CSF, IL-3, and IL-5 receptors are the only receptors known to
transduce signals leading to eosinophil production and, significantly,
the corresponding cytokines can be found concomitantly at elevated
levels in lungs affected by allergic inflammation. The simultaneous
elevation of the GM-CSF, IL-3, and IL-5 receptors may increase
eosinophil numbers, contribute to the overall degree of eosinophil
activation, cause the different phases of eosinophil infiltration, and
determine a localized versus a generalized eosinophil-mediated inflammation.4-6 This may be particularly important in the
pathology of certain diseases, such as asthma, where the eosinophil
plays a major effector role. Thus, an antagonist directed against
c would simultaneously inhibit the function of all 3 eosinophilopoietic cytokines and may prove a useful therapeutic agent.
The extracellular part of c comprises 2 pairs of a
conserved cytokine receptor module (CRM),3 a
membrane-spanning region, and a cytoplasmic domain. Each CRM comprises
2 domains of a fibronectin type III structure with features especially
conserved among cytokine receptors.3 Although
c does not bind cytokines by itself, its coexpression
with the -chains enhances the affinity of cytokine binding, a
process termed affinity conversion. Extensive mutational analysis has
localized the affinity-converting (cytokine-binding) region to residues
in the fourth extracellular domain (D4c) and has shown
that this domain and in particular the residues Tyr365,
His367, Ile368, and Tyr421 within
it are crucial for receptor activation.7-9
A major problem in seeking structural data of the binding site of a
communal subunit complexed to cytokines is that, unlike homodimeric
receptors or isolated -chains of heterodimeric receptors that can
bind directly to cytokines, communal subunits often cannot bind to
cytokines by themselves. We therefore used an antagonistic monoclonal
antibody (mAb), BION-1, which we have shown to reciprocally inhibit
cytokine binding to c10, to form a complex
for crystallographic studies. BION-1, which was raised against
D4c, has been shown to inhibit the high-affinity binding
of GM-CSF, IL-3, and IL-5 to human eosinophils and their production and
functional activation in vitro.10 Within c,
residues Glu366, Arg418, and
Met363 or Arg364 were found to be required for
binding BION-1.10
BION-1 thus represents the first common antagonist of the GM-CSF, IL-3,
and IL-5 receptors, and it is a unique tool with which to explore the
cytokine-binding site in the common c. Here we report
the crystal structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor signaling subunit bound to the mAb antagonist, BION-1. The
structure provides a molecular basis for understanding ligand recognition and receptor assembly. Furthermore, the structure of the
complex provides leads for the design of novel therapeutics against
allergic diseases.
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Materials and methods |
Crystallization and data collection
D4c (residues 338-438 with an additional
amino-terminal Met) was expressed using the pEC611 vector in
Escherichia coli and purified by reverse-phase high-performance
liquid chromatography (HPLC). The expressed protein was insoluble, but
it could be recovered from the bacteria by dissolution in 6 mol/L
guanidine hydrochloride and 50 mmol/L sodium acetate buffer (pH 4.0).
After HPLC the protein was dialyzed exhaustively against 5 mmol/L
2-[N-morpholino]ethanesulfonic acid (MES) buffer (pH
6.0). The BION-1 mAb was raised against D4c,10 and Fab fragments were generated and
purified by standard methods. The complex was produced by mixing BION-1
Fab and D4c to give a 1:1 (mol/mol) complex, which was
purified on a gel filtration column (Superdex 75; Amersham Pharmacia,
Little Chalfont, England).
Crystals of the complex were grown by the hanging-drop vapor diffusion
method at 22°C. We mixed 2-µL droplets of protein solution (protein concentration of 5-7 mg/mL) with 1.5 µL of the reservoir solution. The solution was then equilibrated against a 1-mL reservoir consisting of 100 mmol/L citrate buffer (pH 5.5) containing 12% (wt/vol) polyethylene glycol 4000. The crystals reached maximum size of
approximately 0.6 mm × 0.2 mm × 0.2 mm over 10 days.
The crystals belonged to space group P41 212
and had the following cell dimensions: a and
b were 7.76 nm, and c was 29.49 nm. The crystals were
micromanipulated, washed several times in reservoir buffer, and
dissolved in sodium dodecyl
sulfate-N-tris[hydroxymethyl]methylglycine (SDS-Tricine) sample buffer. Polyacrylamide gel electrophoresis (PAGE)
was performed to confirm that the crystals were of the intact complex.
The crystals proved to be sensitive to radiation, and hence,
cryocooling was essential. However, they were fragile, and an array of
commonly used cryoprotectants caused disordering of the crystals. A
flash-freezing protocol was finally established. This involved soaking
the crystals in 5% (vol/vol) increments of 2-methyl-2,4-pentanediol
for 2 minutes to a final concentration of 15% (vol/vol).
A native data set was initially collected in-house on an imaging plate
area detector (MARResearch, marUSA, Evanston, IL) with Cu
K x-rays generated by a rotating anode generator
(Rigaku RU-200, Molecular Structure Corp., The Woodlands, TX). A better
native data set was subsequently collected from a single crystal frozen to -273°C using synchrotron radiation (BioCARS beamline, 14-BM-C; Advanced Photon Source, Chicago, IL). The diffraction data were processed and analyzed using DENZO and SCALEPACK11 and
programs in the CCP4 suite12 (Table
1).
Structure determination
The crystal structure was solved by molecular replacement using
AmoRe14 and the in-house native data set.
Nonredundant Fab fragments were downloaded from the protein databank
(PDB) and systematically tested as molecular replacement search probes. The second search probe tested, a mouse Fab fragment with PDB identifier 1YEC,15 proved successful. The 10th peak in the rotation function (peak height of 3.3  ) produced the highest peak in
the translation function (with a correlation coefficient of 27.9 and an
Rfactor of 54.1% compared with the next highest peak, which had a correlation coefficient of 17.3 and an
Rfactor of 57.5%). The statistics indicated that
P41 212 was the correct enantiomorphic space
group. Rigid body refinement of the initial solution lead to a model
with a correlation coefficient of 28.7 and an
Rfactor of 49.9% (resolution range, 1.0-0.45 nm). Further refinement, in which the Fab domains were treated as separate rigid bodies, resulted in further improvement of the statistics (an
Rfactor of 46.1% and a drop of
Rfree from 50.9% to 43.8%). Maps calculated from
this solution yielded readily interpretable density for
D4c.
The model of the complex was then built with the help of skeletonized
maps using the program O16 and refined using
the maximum likelihood target in the program package CNS.17
The refinement was completed with the synchrotron native data set
(Table 1). In the final stages a bulk solvent correction and restrained
individual isotropic B-factors were applied. The quality of the final
map was very good, with no breaks in the main-chain connectivity, and
the real space fit16 of residues into the map never fell below 0.7. The final model comprises residues 338-438 for
D4c; all residues for the Fab fragment; 124 solvent molecules; and 1 carbohydrate unit, an N-acetylglucosamine
unit off the BION-1 residue Asn26L.
The choice of solvent molecules was conservative. The molecules were
only accepted if they appeared as peaks, with a signal of more than 3 times the SD error in difference maps; reappeared in subsequent
2Fo-Fc maps; took part in at
least 1 hydrogen-bonding interaction; and had temperature factors of
less than 0.8 nm2. The stereochemical quality of the final
model is good (Table 1), and other stereochemical parameters, such as
side-chain chi angle values, peptide bond planarity, alpha-carbon
tetrahedral distortions, and nonbonded interactions, are all
significantly better than the ranges allowed according to the program
PROCHECK.13 The correctness of the tracing is
supported by residue omit maps in which 10% of the model was deleted,
a round of simulated annealing was performed to reduce bias, and the
resultant map was examined in the region of omission. The tracing is
also supported by 3D-1D scores that never fall below 0.2, which
indicates that there are no residues in chemically unreasonable
environments.18
Functional studies
Human Embryonal Kidney 293T (HEK293T) cells were grown in RPMI-1640
(GIBCO Laboratories, Glen Waverly, Vic., Australia) containing 10%
fetal calf serum (FCS) and were cotransfected with expression constructs encoding IL-3 receptor -chain, c, and Janus kinase (JAK-2) using a calcium-phosphate precipitation procedure. Briefly, 1.4 × 106 cells were plated onto 6-cm plastic
tissue culture dishes the day before transfection and left to adhere
overnight. Four hours after a medium change, 10 µg DNA was added in
the form of a calcium phosphate precipitate, which was left on the
cells for a further 4 hours. The expression constructs used per
transfection were 6 µg wild-type or mutant c
complementary DNA (cDNA) cloned into pcDNA1 (Invitrogen, Groningen, The
Netherlands), 3 µg IL-3 receptor -chain cDNA in pCDM8, and 1 µg
JAK-2 in pRcCMV (Invitrogen). The cells were then released from the
plates, replated in flasks, and incubated for a further 40 hours before
use in an activation assay. On the day of the functional experiment,
the cells were released and washed in cold phosphate-buffered saline
(PBS) and subsequently stimulated with IL-3 at the concentration
specified for 5 minutes on ice. Lysates were prepared, precleared and
immunoprecipitation was carried out with an anti-c mAb,
8E4, essentially as described previously.19 After extensive
washing, immunoprecipitates were separated on a 7.5% SDS-PAGE gel
under reducing conditions, transferred to nitrocellulose, and
immunoblotted with antiphosphotyrosine antibody, PY20 (Transduction
Laboratories, Lexington, KY). The blot was developed by enhanced
chemiluminescence (ECL) (Amersham, Pharmacia) and then stripped and
reprobed with anti-c antibody 1C1 to control the amount
of c present.
Epitope mapping
African green monkey COS cells were maintained in RPMI with 10% FCS
and transfected with 10 µg of wild-type or mutant c
expression construct by electroporation essentially as described
previously.8 The cells were washed in PBS and lysed as
detailed elsewhere19 48 hours after transfection. Lysates
were run on a 7.5% SDS-PAGE under reducing conditions, and
immunoblotting was carried out after electro-blot
transfer using either the antagonistic mAb, BION-1, or
anti-c mAb, 1C1, at 1 µg/mL. This was followed by the
addition of antimouse Ig coupled to horseradish peroxidase (Pierce,
Rockford, IL). Blots were developed by ECL (Amersham) following
manufacturer's instructions.
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Results |
Crystal structure of the GM-CSF/IL-3/IL-5 receptor c
activation domain
We expressed D4c in E coli and purified it to
homogeneity by reverse-phase HPLC. BION-1 mAb was digested with ficin
to generate Fab fragments that were purified by chromatography on
protein A sepharose. Titration of D4c and the BION-1 Fab
produced a stoichiometric 1:1 complex that subsequently formed
crystals. These crystals diffracted well enough to allow a full
crystallographic structure determination to proceed.
We determined the structure of the BION-1/D4c complex to
a resolution limit of 0.28 nm. The structure showed that the
D4c molecule has a compact globular shape with overall
dimensions of 4.5 nm × 2.5 nm × 2.0 nm (Figure
1). The amino-terminus and carboxy-terminus
represent the sites of attachment for the remainder of the
extracellular region and the membrane-spanning domain, respectively.
The molecule adopts the topology of a fibronectin type III module with
2 antiparallel -sheets (42% sheet) packing against each other via a
multitude of hydrophobic interactions including 2 clusters of aromatic
residues Trp434, Tyr354, and
Tyr376; Trp358, Phe372, and
His370). Sheet A consists of 3 beta-strands: strand A is
comprised of residues 344-350, B of residues 353-359, and E of residues
396-398. Sheet B consists of 4 strands: strand C is comprised of
residues 369-378, D of residues 389-392, F of residues 406-417, and G
of residues 432-436). The longest strand, F, almost spans the entire length of the molecule.
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| Fig 1.
Structure of D4c.
(A) Structure of the Fab receptor D4c complex shown in
ribbon representation. The mAb light chain is shown in cyan blue, the
heavy chain in blue, and the receptor in yellow. The major structural
features of D4c are labeled, and the locations of key
residues are denoted by stick representation. These pictures were
produced using the Molscript20 and Raster3D21
programs. (B) Structure as for (A) but reoriented 90°
about the vertical axis. (C) Surface representation of the receptor
using the program GRASP.22 The green surface indicates the
location of hydrophobic-aromatic patch H1. The molecule is tilted
approximately 20° counterclockwise relative to (A). (D) View of
hydrophobic-aromatic patch H2 prepared as for (C). The molecule is
tilted approximately 20° clockwise and rotated approximately
60° clockwise from above, about a vertical axis relative to (B).
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The amino acid sequence motif WSXWS (tryptophan-serine-any
residue-tryptophan-serine) is a characteristic feature of many cytokine
receptors. WSXWS is located between F and G strands and adopts a double
-bulge structure (Figure 2) with the
tryptophan side chains interdigitated between the arginine side chains
from the adjacent F strand. In D4c, this ladder of
alternating basic and aromatic residues is extended and consists of the
following side chain:
Arg415-Trp425-Arg413-Trp428-Arg411-Trp383-Arg377-Trp409-Arg407.
There is a "sidestep" in the ladder at
Arg377-Trp409. This 9-rung ladder, measuring
2.9 nm long with rungs of about 0.5 nm wide, represents the only
significant electropositive patch on the surface of the molecule.
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| Fig 2.
View of the Trp/Arg "ladder."
Structure of the D4c shown in ribbon representation with
the side chains of the Trp/Arg stack shown as ball and stick. The
molecular graphics were produced using the Molscript20 and
Raster3D21 programs.
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There are 2 large hydrophobic patches on the surface of
D4c: H1 and H2. The first, H1, forms part of a lip at
the end of a pronounced groove on the surface of the molecule (Figure
1C). This patch is made up of residues Ile338,
Met340, Ala341, Pro342,
Met361, Tyr365, the aliphatic moiety of
Lys362, Ile368, and Tyr421. The
groove is located at the N-terminal end of the molecule, where one wall
is formed by the B-C loop and part of the F-G loop, and the other wall
is formed by the N-terminus (residues 338-342) (Figure 1C). The second
hydrophobic patch, H2, located on the opposite face to the first, is a
dense strip of hydrophobic residues located at one edge of the
-sandwich defined by the D and E strands. It measures 2.7 nm × 0.6 nm (Figure 1D).
Of the interstrand loops, only the B-C and F-G loops protrude
significantly from the body of the protein; both have been implicated in cytokine binding.3,7-9 The B-C loop adopts significant
regular structure with residues 365-368, forming a type I -turn
(Figure 1A). Significant features of the B-C loop of D4c
include residues Tyr365 and His367, both of
which project out into the solution (Figure 1A, B). The F-G loop adopts
a type I' -turn at its tip, and the most significant features
in this region are Arg418 and Tyr421,
both of which project away from the body of D4c
(Figure 1A, B).
Functional roles of the B-C loop and Tyr421
Although the structure of D4c revealed that
Tyr421 is in close proximity to the 3 residues in the B-C
loop involved in cytokine binding (Tyr365,
His367, and Ile368), the side chain is oriented
away from these, possibly reflecting different functional roles.
Previous experiments9 suggested that high-affinity binding
of IL-3 was sensitive to mutation of Tyr421 but not to
replacement of individual residues in the B-C loop.8 We
examined whether a multiple mutation in the B-C loop of the residues
implicated in binding GM-CSF and IL-5 would affect IL-3 high-affinity
binding. The results showed that alanine substitution of residues
365-368 in the B-C loop abrogated high-affinity binding of both GM-CSF
and IL-3 (Figure 3A). We next examined
phosphorylation of cytoplasmic tyrosine residues, as this is a very
sensitive measure of recruitment of c to a
ligand/-chain complex. Some analogs of c are unable
to affinity-convert due to the affinity of the - complex for
cytokine being less than or equal to that of the -chain alone. These
same analogs may nevertheless exhibit differences in tyrosine
phosphorylation (see "Discussion"). We examined the effects of
mutating the B-C loop or Tyr421, either separately or in
combination, on the ability of c to undergo tyrosine
phosphorylation in response to IL-3. We found that substitution of
Tyr421 had a pronounced effect, with high levels of
tyrosine phosphorylation of c being achieved only at 3 µmol/L IL-3, a concentration about 500-fold higher than that required
by the native receptor (6 nmol/L). In contrast, mutation of the B-C
loop alone did not impair IL-3-induced phosphorylation of
c at the high concentration used. Nevertheless, a role
for the B-C loop in c activation was demonstrated by a combined mutant of the B-C loop and Tyr421 (Figure 3B),
which abrogated IL-3-induced tyrosine phosphorylation of
c.
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| Fig 3.
Differential effects of mutating the B-C loop and/or
Tyr421 of the F-G loop in receptor activation.
(A) Scatchard plot transformation of binding isotherms
for 125I-GM-CSF and 125I-IL-3 to cells
transfected with wild-type c ( ) or
365AAAA368 mutant c ( ). (B)
Western blot of wild-type and mutant c after stimulation
with various concentrations of IL-3. The blot was probed for
phosphotyrosine (upper panel) and c (lower panel). The
double bands in each lane of the gels represent glycosylation variants
of c.23
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Antagonist interactions with the
c activation domain
A detailed analysis of the structure of the
BION-1/D4c complex confirmed and extended the
observations that BION-1 appeared to form extensive and intimate
interactions with the receptor activation domain (Figure 1A, B and
Figure 4). The total surface area buried on complex formation is 15 nm2, which is in the range reported for other
antibody-protein antigen complexes.24 In total, there are 2 salt bridges (Lys362/AspL94 and
Glu366/LysH35), 8 potential hydrogen
bonds, and 124 van der Waals (vdw) interactions (Table
2). The B-C loop of D4c is
nestled in the shallow antigen-binding groove between the
VH and VL domains, whereas the F-G loop forms a
more peripheral interaction with complementarity determining region one
of the light chain (CDR L1) of BION-1 (Figure 1A, B and Figure 4). The
contact surface comprises 14 residues from BION-1, with 9 residues from
VH and 5 residues from VL. The majority of
contacts are roughly shared between 4 of the CDRs: CDR L1 (1 hydrogen
bond and 29 vdw contacts); CDR L3 (1 salt bridge, 3 hydrogen bonds, and
28 vdw contacts); CDR H1 (1 salt bridge, 3 hydrogen bonds, and 36 vdw
contacts); and CDR H3 (1 hydrogen bond and 23 vdw contacts). In
addition, CDR H2 provides 8 vdw contacts, but CDR L2 makes no contacts
with the receptor domain.
In total, 6 residues from the B-C loop (between residues 362-368) and 3 residues from the F-G loop (between residues 416-422) of
D4c are involved in antibody interactions with those
from the B-C loop, accounting for 75% of the total. The B-C loop
interacts with CDRs H1, H2, H3, L1, and L3, whereas the F-G loop
interacts only with CDRs H3 and L1 (Figure
4). There is one small cavity of 0.0099 nm3 in the antibody-antigen interface. The cavity is lined
by residues Tyr365, His367, and
Ile368 of the receptor and residues Val27,
Tyr28, Phe32, and Asn92 of the
antibody light chain. Not all of the potential salt bridges and
hydrogen bonds identified above are likely to contribute productively to complex formation because substitution analysis has only identified Glu366, Arg418, and Met363 or
Arg364 in D4c as contributing to the epitope
for binding BION-110 (Table 2).
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| Fig 4.
The BION-1/D4c interface.
D4c is shown as a surface representation colored
according to the functional effect of residue substitution. Blue
represents residues whose substitution abrogates binding of BION-1 but
does not affect affinity-conversion. Red represents residues whose
substitution reduces affinity-conversion but does not affect binding of
BION-1. Yellow represents residues whose substitution does not affect
binding of BION-1 or cytokines. Gray represents residues that have not
been examined by mutation and do not contact BION-1. The identities of
key D4c residues are shown in italics. Residues in
BION-1 that contact D4c are shown in stick
representation and colored cyan blue (hydrophilic) or brown
(hydrophobic-aromatic). The backbone atoms of residues colored gray
show the connectivity of the loops.
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Discussion |
We describe here the structure of the activating domain of the
common c of the GM-CSF/IL-3/IL-5 receptors complexed
with the Fab fragment of the antagonistic mAb, BION-1. The structure shows general features typical of the cytokine receptor superfamily as
well as unique features that reveal how a single receptor subunit can
interact with 3 different cytokines. Functional analyses show the
separate but cooperative interplay of the B-C loop and
Tyr421 in the F-G loop in receptor activation.
A number of related (class 1) cytokine receptor structures are known:
growth hormone receptor (GHR),26 prolactin receptor (PRLR),27 erythropoietin receptor (EPOR),28
G-CSF receptor (G-CSFR),29 gp130,30
and the IL-4 receptor -chain (IL-4R-chain).31 The
pair-wise sequence identities between D4c and these
receptors, after structure-based alignment, range from 12%
(G-CSF) to 27% (gp130). There are only 7 residues
(Pro343, Trp358,
Leu402, Tyr408, Arg413,
Gly423, and Ser426) that are strictly conserved
across the receptors; all appear to play structural roles. The
structural importance of Trp358 is highlighted by the
observation that its substitution or the substitution of neighboring
Tyr356 by Asn abrogates affinity conversion by
c.32 A structural superposition indicates
that D4c is most closely related to PRLR (0.16 nm
studies on 88 C atoms, 20% sequence identity) followed by GHR (0.19 nm on 81 C atoms, 23% sequence identity). The root mean square
deviation of other receptors indicates that (1) the membrane-distal B-C
loop of the second domain within a CRM is normally involved in
cytokine-binding and (2) the neighboring F-G loop of this domain and
the A-B and E-F loops of the first domain also make contributions to
cytokine-binding. The B-C loop of D4c, in particular
Tyr365 and His367, has been found to be
involved in cytokine binding (Figure 3).7,8 G-CSFR, GHR,
PRLR, and IL-4R-chain have an aromatic residue in an equivalent
position to Tyr365, whereas only the IL-4R-chain has an
aromatic residue (tyrosine) similar to His367 of
c. The IL-4R-chain is also the only receptor of known
structure that has an aromatic residue (tyrosine) equivalent to
Tyr421 in the F-G loop.
The most salient features of the D4c crystal structure
are the 2 hydrophobic-aromatic patches and the distinct groove, which is formed in part by the B-C and F-G loops and hence located at the
putative cytokine-binding site. The hydrophobic-aromatic surface patches, H1 and H2 (Figure 1C, D), have corresponding features in most
of the other receptors. With the exception of gp130, all the receptors
possess significant hydrophobic-aromatic patches equivalent to the
location of H2 (centered about the D-E strand connection), although the
degree and extent of hydrophobicity varies greatly. The corresponding
H2 patch of GHR (Figure 5) forms part of the surface
involved in subunit contacts.26 This is suggestive of a
role for the H2 of D4c in association with -chains, particularly the GMR with which it associates
spontaneously.23 The equivalent region to H1 is conserved
in all but gp130. By analogy with the other receptors, the H1 patch of
D4c might interact with the A-B loop from domain 3 of
the intact receptor. The groove is only present in G-CSFR, whereas the
N-terminal ends of the equivalent domains of EPOR and gp130 are rather
flat, and those of GHR and PRLR are mostly flat, with the exception of
a tryptophan residue that protrudes into the solution.
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| Fig 5.
Comparison of D4c with the
membrane-proximal domain of GHR.
D4c and domain 2 of the subunit of the GHR, which
interacts with the helix A/helix C face of GH, were aligned
structurally via their core residues and are shown as surface
representations using the program InsightII (MSI, San Diego, CA). The
hydrophobic-aromatic patch, H2, of D4c and the location
of GHR that interacts with the opposing receptor molecule are indicated
by green surfaces. The red surfaces of D4c indicate the
residues required for affinity-conversion, and the blue surfaces of GHR
indicate the region known to interact with GH.
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There are considerable amounts of mutagenesis data available that
indicate which regions of the receptor and the cytokine interact with
each other. In the cytokines there is an essential glutamate
(Glu21 of GM-CSF, Glu22 of IL-3, and
Glu13 of IL-5) involved in binding to
c.33-35 The loops of domains 3 and 4 of
c have been the subject of extensive mutagenesis that has led to the following conclusions: (1) Tyr365,
His367, and Ile368 of the B-C loop are
implicated in cytokine interaction, whereas mutations of other residues
in this loop have little or no effect on binding.7,8
Substitution of any of these residues by alanine led to a loss of
affinity-conversion of GM-CSF and IL-5 binding,8 whereas
the Phe365 mutant retained affinity-conversion of GM-CSF
binding.7 (2) The major residue in the F-G loop that has
been implicated in binding9 is Tyr421. (3) To
date, there have been no residues in domain 3 implicated in
binding.3
The crystal structure of D4c provides a molecular
explanation of how each cytokine, with less than 15% pair-wise
sequence identity, can recognize the signaling subunit. The side-chains of the 3 key residues in the B-C loop that interact with a cytokine, as
identified by the mutagenesis studies, are seen to converge closely at
their tips (Figure 1A). Thus Tyr365, His367,
and Ile368 may play a pivotal role by promoting an
interaction with the essential glutamate residue in all 3 cytokines.
This may involve formation of a hydrogen bond between the glutamate
residue and Tyr365 or His367 or the cooperative
formation of part of the cytokine-binding surface. In this context it
is worth noting that in the IL-4 receptor system, the homologous
Tyr127 is close to Glu9 but does not form a
hydrogen bond with it.31 All other residues of the B-C loop
are orientated in a different direction from the binding triad, which
is consistent with their not taking part in binding cytokines. On the
other hand, Tyr421 in the neighboring F-G loop is
positioned to contribute directly or indirectly to binding cytokines.
Directly, through its hydroxyl group, Tyr421 may form a
hydrogen bond with the conserved glutamate. This would be akin to the
known interaction of Tyr183 in a homologous position in
IL-4R-chain and Glu9 of IL-4.31 Indirectly,
Tyr421 may interact with the A-B loop of domain 3 of
c, as seen with Phe205 of
EPOR,28 and thus support an appropriate orientation of this domain, or Tyr421 may facilitate receptor assembly. Given
that the active receptor probably has a stoichiometry of 2 -chains :
2 c : 2 ligands, Tyr421 may directly
interact with either a second c subunit or -chain subunit in the hexameric complex.3
The separate and combined mutagenesis of the B-C loop and
Tyr421 revealed that both sites are involved in
high-affinity binding and receptor activation of all 3 cytokines,
GM-CSF, IL-3, and IL-5, albeit in subtly different ways. The
observation that the tetra-alanine substitution of the B-C loop
abrogated IL-3 high-affinity binding (Figure 3A) is particularly
interesting because single or paired alanine substitutions along this
loop have marginal or no effect on IL-3 high-affinity
binding.8 The latter is in contrast to GM-CSF and IL-5,
where substitution of either Tyr365, His367, or
Ile368 completely eliminates high-affinity
binding.7,8 Conversely, substitution of Tyr421,
while abrogating high-affinity binding of all 3 cytokines, has a
profound effect on IL-3 receptor activation, as measured by phosphorylation of cytoplasmic tyrosine residues of c
(Figure 3B), but a minor effect on the activation of the GM-CSF
receptor.9 These differences in receptor activation
probably reflect the different abilities of mutated c to
be recruited to IL-3/IL-3R-chain complexes and suggest that, in the
case of Y421A, the stability of the active
IL-3/IL-3R-chain/c complex is considerably lower than
even that of an IL-3/IL-3R-chain complex.
The combination of substitutions within the B-C loop and
Tyr421 caused a complete loss of tyrosine-phosphorylation
of c. This suggests that even in the presence of
concentrations of IL-3 that saturate the IL-3R-chain,
c is not recruited significantly (Figure 3B). These
functional observations in the context of the structure support the
notion of a central cytokine-binding "hot spot" in D4c, with a structural plasticity that allows it to
accommodate 3 cytokines of significant diversity as well as monomeric
(GM-CSF and IL-3) and dimeric (IL-5) structure. In this model, GM-CSF may interact more closely with the B-C loop, while the orientation of
IL-3 may be slightly different and more dependent on Tyr421
for its interaction with c. Comparison of the
cytokine-binding surface of D4c with the corresponding
surface of GHR reveals substantial similarity in terms of the parts of
the B-C and F-G loops involved (Figure 5), although the contributions
to cytokine binding of these GHR residues have not been assessed by
mutational analysis for this homodimeric receptor. The location of
these cytokine-binding residues of c relative to the
hydrophobic patch, H2, which may interact with -chains, is similar
to the intermolecular contacts seen in the GH:GHR
complex.26 Ultimately, solving the structures of the GM-CSF
and IL-3 receptor complexes may provide a definitive answer to the
relative positioning of GM-CSF, IL-3, and their -chains in respect
to c.
The epitope of D4c that interacts with cytokines,
largely overlaps the surface that is recognized by BION-1. Although
several residues that are required for affinity-conversion
(Tyr365 and His367 and others such as
Lys362 make intimate contact) with BION-1, they are not
required for binding of the mAb. Rather, a set of adjacent residues,
including Met363 or Arg364, Glu366
and Arg418, provides the key determinant for binding BION-1
(Figure 4). While the basis for the roles of these residues can be seen
clearly from the structure, the absence of productive contributions to binding from other residues, especially Tyr365 and
His367, suggests that their corresponding contacts in
BION-1 may be targets for mutagenesis. This may lead to improved forms
of the mAb or derivatives of it, which may be higher affinity
antagonists. Because BION-1 has been shown to inhibit the
GM-CSF/IL-3/IL-5-induced proliferation of eosinophils in
vitro,10 this highlights the possibility of developing
single-molecule antagonists of several cytokines. This approach of
targeting a common receptor subunit may also be extended to other
receptor chains, such as the common subunit of the IL-4/IL-13
receptors, which mediates allergen-induced asthma induced by IL-4 and
IL-13.36
Note added in proof: The coordinates have been deposited in the
Research Collaboratory for Structural Bioinformatics Protein Data Bank,
code 1EGJ.
| |
Acknowledgments |
We thank Craig Gaunt, Betty Zacharakis, and Frosa Katsis for technical
assistance and Elspeth Garman for advice on flash freezing of crystals.
We also thank Harry Tong and other staff at BioCARS for their help with
data collection during our visit to Advanced Photon Source.
| |
Footnotes |
Submitted September 17, 1999; accepted December 15, 1999.
Supported in part by a grant from the National
Health and Medical Research Council of Australia, Canberra, Australia,
and a grant from the Australian Synchrotron Research Program,
Australian Nuclear Science and Technology Organisation, Menai,
Australia, which is funded by the Commonwealth of Australia under the
Major National Research Facilities Program, Australia. Use of the
Advanced Photon Source was supported under contract W-31-109-Eng-38
from the U.S. Department of Energy, Basic Energy Sciences, Office of Science, Washington DC. Use of the BioCARS Sector 14 (Advanced Photon
Source, Argonne National Laboratory, 9700 South Cass Ave, Argonne, IL)
was supported by grant RR07707 from the National Institutes of Health,
National Center for Research Resources, Bethesda, MD.
J.R. and W.J.M. contributed equally to the structural
biology aspects of this work. J.R. is a postdoctoral fellow of the
Australian Research Council, Canberra, Australia. J.M.W. is a fellow of
the Anti-Cancer Foundation of South Australia, Adelaide, South
Australia, Australia; M.W.P. is a senior research fellow of the
Australian Research Council, Canberra, Australia; and C.J.B. is a
Florey fellow of the Royal Adelaide Hospital, Adelaide, South
Australia, Australia.
Reprints: Angel F. Lopez, Cytokine Receptor
Laboratory, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Rd, Adelaide, South Australia 5000, Australia; e-mail: angel.lopez{at}imvs.sa.gov.au; or Michael W. Parker, The Ian Potter Foundation Protein Crystallography Laboratory, St
Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy,
Victoria 3065, Australia; e-mail: mwp{at}rubens.its.unimelb.edu.au.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction