Functional epitope of common c chain for interleukin-4 binding
Jin-Li Zhang, Manfred Buehner and Walter Sebald
Theodor-Boveri-Institut fu
¨
r Biowissenschaften (Biozentrum), Physiologische Chemie II, Universita
¨
tWu
¨
rzburg, Germany
Interleukin 4 (IL-4) can a ct on target cells through an IL-4
receptor c omplex consisting of the IL-4 receptor a chain and
the c ommon c chain (c
c
). A n IL-4 epitope for c
c
binding has
previously been identified. In this study, the c
c
residues
involved in IL-4 binding were defi ned by alanine-scanning
mutational analysis. The epitope comprises c
c
residues I100,
L102, and Y103 on loop EF1 together with L208 on loop
FG2 as the major binding determinants. These predomin-
antly h ydrophobic determinants i nteract with t he hydro-
phobic IL-4 epitope composed of residues I11, N 15, a nd
Y124. Double-mutant cycle a nalysis revealed co-operative
interaction between c
c
and IL-4 side chains. Seve ral c

system is a p rerequisite for the rational design of IL-4-like
drugs.
IL-4 is one of the s hort-chain four-helix bundle cytokines.
Its effects depend on binding to and s ignaling t hrough a
receptor complex consisting of a p rimary high-affinity
binding subunit, the IL-4Ra, a nd a l ow-affinity receptor,
depending on the cell type, the common c chain (c
c
;typeI
IL-4 receptor [8]) or IL-13Ra1 chain (type II IL-4 receptor
[9]). All three receptors are members of the type I cytokine
receptor superfamily, w hich is charac terized by the presence
of at least one cytokine-binding homology r egion (CHR)
composed of two fibronectin type III domains. The
membrane distal domain contains a set of four conserved
cysteines, and the membrane proximal domain contains a
WSXWS motif [10]. T he fibronectin type III domain is
comprised of s even b strands, t he sequences of which are
conserved b etween members o f the family, while loop
sequences connecting the b strands vary between family
members and putatively contain r esidues that mediate
distinct intermolecular c ontacts. These loop regions were
therefore selected for this mutational analysis.
A comprehensive mutational analysis of I L-4 i n w hich
single residues were replaced by alanine or charged residues
yielded high-resolution data on the binding epitopes f or the
receptor chains. The IL-4 site 1 binding epitope for IL-4Ra
consists of a mixed charge pair (E9, R88) as major
determinants and five minor determinants l ocated on helices
A, B, and C [11]. The importance of site 1-binding

t
Wu
¨
rzburg, Am Hubland, D -97074 Wu
¨
rzburg, Germany.
Fax: + 49 931 888 4113, Tel.: + 49 931 888 4111,
E-mail:
Abbreviations: IL-4, interleukin-4; IL-4Ra, interleukin-4 receptor a
chain; IL-4BP, IL-4 binding protein; c
c
, common c chain; IL-13Ra1,
IL-13 receptor a1 chain; CHR, cytokine-binding homology region;
Jak, Janus kinase; XSCID, X-linked severe combined immunodefi-
ciency; hGHR, human growth hormone receptor; hEPOR, hum an
erythropoietin receptor; b
c
,commonb chain.
(Received 14 November 2001, revised 16 January 2002, accepted
21 January 2002)
Eur. J. Biochem. 269, 1490–1499 (2002) Ó FEBS 2002
c
c
is shared by several important cytokine receptor
complexes, including those for IL-2, IL-4, IL-7, IL-9, IL-15
[8] and also for the recently described new member of the
cytokine family, I L-21 [19]. c
c
alone binds ligands with very
low affinity (K

So far, the binding epitopes o f human and m urine c
c
for some c
c
-dependent cytokines have been studied.
A molecular mapping study using the antagonistic
monoclonal a ntibody PC.B8, which reacts with a discon-
tinuous site on human c
c
, localized c
c
binding residues to
four loops, but did not identify single specific residues for
ligand binding [29]. Mutational analysis of murine c
c
employing heterodimeric IL-2R and IL-7R on whole cells
suggests that c
c
epitopes for IL-2 and IL-7 binding
overlap a nd comprise at least t hree distinct putative loop
segments of the c
c
protein [ 30]. Here we report the effect
of single amino-acid substitutions in the human c
c
ectodomain on IL-4 binding. Biosensor techniques
employing s oluble r ecombinant I L-4, IL-4-BP and the
wild type or mutant forms of human c
c
ectodomain

residues 1–233 [31] was cloned into the temperature-
regulated e xpression vector pRpr9 fd [32], expressed i n
Escherichia coli strain KS 474, and refolded as desc ribed
[33]. The refolded protein was purified to homogeneity by
gel-filtration chromatography through a Superdex 200 HR
10/30 column, and stored at )80 °C.
The A 182, C207 IL-4BP variant was produced in SF9
cells, purified, and biotinylated at C207 as described [32].
IL-4 and IL-4 variants were expressed in E. coli, refolded,
and pu rified to homogeneity as described [11,34]. Protein
concentrations were determined by measuring A
280
,using
an absorption coefficient (e
280
) ¼ 8860
M
)1
Æcm
)1
for
IL-4, e
280
¼ 7370
M
)1
Æcm
)1
for A124 IL-4, e
280

)1
Æcm
)1
for murine c
c
.
Mutagenesis of the c
c
ectodomain
cDNA for human c
c
ectodomain was submitted to in vitro
cassette mutagenesis employing synthetic double-stranded
oligonucleotides. The c
c
variants were expressed and
purified as the wild-type human c
c
ectodomain.
Biosensor interaction analysis
The binding of c
c
variants to I L-4/IL-4BP was recorded on a
BIAcore 2000 system ( Pharmacia B iosensor) as described
[15]. Briefly, a CM5 biosensor chip was first loaded with
streptavidin in flow cells 1 and 2. Subsequently biotinylated
A182,C207 IL-4BP was immobilized at the streptavidin
matrix of flow cell 2 at a density of % 200 resonance units.
The following reaction cycle was applied using the c om-
mand COINJECT: ( a) IL-4 at 0.1 l

binding of c
c
variants at 1, 2, 3, 5, 10 l
M
was measured for at
least three times in duplicate. The mean standard deviation
(mean r) was 13.8% ± 6 .5% for the K
d
values calculated
from the fi ve variant concentrations. For the double mutant
cycle analysis [35], the same procedure as a bove was used
except that IL-4 variants [15,36] at 0.1 l
M
and c
c
variants at
2, 4, 6, 10, 20 l
M
were perfused (the mean r was
16.4% ± 7.4% for the K
d
values). The loss of binding free
energy on mutatio n for IL-4 and c
c
wascalculatedasddG
(kJÆmol
)1
) ¼ 5.69 log K
d
(mutant)/K

¼ 0indicatesthat
the pair o f residues analyzed do n ot interact. A positive
value of ddG
int
means that two residues interact favorably,
and a negative value means that the two residues repel each
other [37]. The individual errors (2 r, a ¼ 0.95) calculated
from the mean for ddG
int
areshowninTable3.
Ó FEBS 2002 Mutagenesis of human c
c
ectodomain (Eur. J. Biochem. 269) 1491
Molecular modeling of the IL-4–IL-4BP–c
c
ternary
complex
The present model i s based on the crystal structure of the
complexofIL-4andIL-4BP(PDBentry1IAR[12]),
augmented by the model of c
c
derived f rom human growth
hormone receptor (hGHR), as obtained from an older
model (T. Mueller, & W. Kammer, personal communica-
tion, Universita
¨
tWu
¨
rzburg, Germany)
1

could be
transferred to the new m odel without major problems.
The local program
DISDM
2 was used (H. J . Hecht, &
M. Buehner, unpublished r esults) t o build and adjust the
present model using the data of mutational a nalysis of IL-4
and c
c
. The program runs under Open-VMS and uses
Datagraph VTC 8002 and VTC 8003 terminals for display.
All model building was performed manually. For online
refinement of conformational energy, the p rogram
EREF
was used [43], which is called from within
DISDM
2.
RESULTS
Site-specific mutagenesis of amino acids
in the c
c
ectodomain
Alanine substitutions were targeted to residues in four
putative i nterconnecting loops and the interdomain segment
of the human c
c
ectodomain based on the published models
[44–46], and sequence alignment performed between c
c
and

ectodomain
expressed i n E. coli occurred as a monomer (Fig. 2). Initial
biosensor studies showed that the different forms of human
and m urine p roteins exhibited similar b inding affinity for
the IL-4–IL-4BP complex. The mixture of monomeric and
dimeric human c
c
interacts with the complex with a K
d
of
Fig. 1. Amino-acid substitutions in the ectodomain of the human com-
mon c chain (c
c
). The amino-acid sequence of c
c
is shown with boxed
portions i ndicating predicted b-strands which are designated by the
letter below the box. R esidues substituted in this study are indicated by
asterisks.
Fig. 2. Gel-filtration analysis of the human c
c
ectodomain expr essed in
SF9 cells and the m urine c
c
ectodomain expressed in E. coli. The samples
were applied to a Superdex 200 HR 10/30 c olumn and eluted w ith the
same buffer. The two peaks of human c
c
represent dimer (A) and
monomer (B).

d
evaluated from the concentration
dependence of e quilibrium binding proved to be very
reliable for measuring the interaction of the c
c
ectodomain
with IL-4–IL-4BP. The measured K
d
for interaction of c
c
ectodomain variants with IL-4–IL-4BP are compiled in
Table 1. Eight c
c
variants including c
c
CHR exhibited
unchanged binding characteristics. Changes in binding
affinity were observed in 11 c
c
variants. The K
d
of six
variants was too high to be r eliably determined. A r ough
estimate yields K
d
values of about 200–300 l
M
for I100A,
L102A, Y103A and L208A, and K
d

The five minor residues investigated contribute only 2.9–
3.5 kJÆmol
)1
. The two cysteine variants C160A and C209A
exhibited a largely r educed binding affinity ( K
d
> 500 l
M
).
Thismaybecausedbystructuralperturbationofthe
protein. A direct role in binding, however, cannot be
excluded for these residues.
Double-mutant cycle analysis of the IL-4–c
c
interface
The c o-operativity of the interaction of some residues on the
IL-4 site 2 epitope and the c
c
ectodomain was determined in
this experiment. Double-mutant cycles were con structed
only for the mutants with minimal effects on b inding
(Tables 2 and 3), because the K
d
values for the interaction
between variants o f the main binding residues I100, L102,
Y103, a nd L2 08 of c
c
andIL-4variantsaswellasthe
interaction betwee n variant I 11A o f I L-4 a nd c
c

c
ecto-
domains w e re applied. Perfusion with buff er alone starte d at time
240 s. The ruler indicates resonance units (RU) corresponding to
1–10 l
M
murine c
c
ectodomain. Th e resonance u nit f or 5 l
M
human c
c
corresponded to that for 1.6–2 l
M
murine c
c
.
Table 1. Equilibrium binding between c
c
ectodomain mutants and
IL-4–IL-4BP. The dissociation constants K
d
were evaluated from
equilibrium binding between wild-type (wt) or mutants (mut) of the c
c
ectodomain and immobilized IL-4BP saturated with IL-4. The l oss of
free energy of binding on mutation was calculated as ddG
(kJÆmol
)1
) ¼ 5.69 log K

N44A 2.8 0.7 )0.9
V45A 3.3 0.8 )0.5
Loop 3 (EF1)
E99A 5.5 1.4 0.8
I100A >320 >80 >11
L102A >240 >60 >10
Y103A >300 >80 >11
Q104A 5.6 1.4 0.8
Loop 4 (ID)
Q127A 2.4 0.6 )1.3
N128A 15 3.7 3.2
Loop 5 (BC2)
N158A 1.4 0.4 )2.3
H159A 17 4.1 3.5
C160A >900 >230 >13
L161A 13 3.2 2.9
E162A 13 3.2 2.9
Loop 6 (FG2)
P207A 3.4 0.9 )0.4
L208A >166 >40 >9
C209A >490 >120 >12
G210A 15 3.7 3.2
Ó FEBS 2002 Mutagenesis of human c
c
ectodomain (Eur. J. Biochem. 269) 1493
G210. The IL-4 side c hain of N15 functionally interacts with
the central receptor s ide chain N128, a nd also with H159
located at the periphery of the functional c
c
epitope. The

was adapted to achieve a good fit to
the core structure (the binary complex; Fig. 5). The
procedure s tarted with moving the whole chain (Ôrigid
bodyÕ). Then domains and subdomains were moved indi-
vidually. The binary core complex was changed as little as
possible, being an experimentally determined structure and
thus the most reliable p art of the model, but some minor
changes in side chain o rientation could not be avoided for
proper a daptation. An important point was to keep t he
C-terminal domains of the receptor c hains c lose together, as
this was expected to be essential for dimer formation and
thereby signaling through the membrane. The structures of
c
c
and the ternary complex were modeled so that residues
that exhibit positive coupling energies during double-
mutant cycle analysis w ere placed c lose to each other.
Occasionally, however, t here was a Ôconflict of interestÕ
between the requirements of interaction and those of
dimerization.
DISCUSSION
This mutational analysis defines human c
c
residues involved
in IL-4 binding. T he residues are located in the EF1, BC2,
and FG2 loops and the interdomain segment of c
c
.The
functional b inding epitope of c
c

d
(mut)/K
d
(wt). ddG
sum
is the sum of th e l osses of f ree
energy of binding upon m ut ation for IL-4 and c
c
separately. ND,
Sensogram could not be evaluated because of weak binding.
IL-4
variants
c
c
chain
variants
K
d
(l
M
)
ddG
(kJÆmol
)1
)
ddG
sum
(kJÆmol
)1
)

Table 3. Co-operativity between r esidue pairs in the i nteraction interface o f c
c
and IL -4. The couplin g e nergy between a p a ir of residues was c alc ulated
as d dG
int
¼ ddG
sum
) ddG (data from Table 2) ac cording to eqn ( 1). T he underlined values indicate f avorable i nteraction. The numbers in
parentheses are the calculated errors (2 r, a ¼ 0.95). ND, Sensogram could not be evaluated because of weak binding.
c
c
chain
variants
ddG of IL-4 variants (kJÆmol
)1
)
N15A R121A Y124F S125A
N128A
1.9 (0.62) )0.2 (0.66) 0.4 (0.80) 1.2 (0.90)
H159A
0.8 (0.64) )0.9 (1.10) )0.3 (0.83) )1.2 (0.86)
L161A )0.3 (0.99)
1.0 (0.80) )0.2 (0.64) )1.6 (0.72)
E162A ND )2.9 (0.90) )3.2 (0.76) )0.7 (0.68)
G210A 0.4 (0.66) )1.4 (0.87) )0.3 (0.48)
0.9 (0.67)
1494 J L. Zhang et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Fig. 5. M odel of I L-4–IL-4BP–c
c
ternary complex. The s tructures o f I L-4, IL-4BP, and CHR of c

). The data for
I11, K12, an d Y124 wer e taken from Letzeler
2
et a l. [15]. The letters in parentheses in (C) indic ate the other mutations found in the s ame p osition .
The figure was produced with
MOLSCRIPT
and
RASTER
3
D
.
Ó FEBS 2002 Mutagenesis of human c
c
ectodomain (Eur. J. Biochem. 269) 1495
better suited to form crystals of IL-4–IL-4BP–c
c
than the
complete c
c
ectodomain for solving the structure of t he low-
affinity complex by X-ray diffraction.
It appears that binding of c
c
to IL-4 is sustained
predominantly by h ydrophobic i nteractions. O f t he nine
residues involved in IL-4 binding, five, in particular all four
major d eterminants, are hydrophobic. We propose that
residues I100, L102, and Y103 of loop EF1, a nd L208 of
FG2 form a hydrophobic c luster to interact with the
hydrophobic epitope composed of residues I11, N15, a nd

and > 90 0 l
M
, respectively).
The two cysteines may form a disulfide bond between loops
BC2 and FG2. This prediction is consistent with our model
and one of the published models [46] of c
c
. The contribution
of the two residues to binding could not be directly
determined. The disulfide bond may be only important for
maintaining the structural integrity of c
c
. However, i t
cannot be ruled out that the disulfide group participates
directly in binding. These questions may be answered when
the structures of both free c
c
and the IL-4–IL-4BP–c
c
ternary complex are solved.
Double-mutant cycle analysis could identify co-operativ-
ity between two side chains [ 35], and predict a more d etailed
map of interacting residues without knowledge of t he
structures of the two proteins analyzed. Unfortunately, the
coupling e nergies between the m ajor determinants on c
c
and
IL-4 site 2 cannot be measured because of the low b inding
affinity of the alanine variants. Nevertheless, our experiment
revealed favorable interactions between several pairs of c

c
could not be exclud ed. Therefore, it would be interesting
to determine t he IL-1 3Ra1 epitope for IL-4 binding and
compare it with the c
c
epitope defined in this experiment.
It is unfortunate for our modeling process that the most
effective mutations did not yield interaction data. Therefore,
we had to rely on the residues of the weaker (but
measurable) interaction which, although they are expected
to work over larger distances and t hus provide less stringent
constraints than d esirable, nevertheless l ead to a quite
reasonable model as far as the gross features are concerned.
For all the details on a t ruly atomic scale, howeve r, we have
to await the crystal structure of the ternary complex.
This study focu ses o n t he molecular description of the
mechanism of recognition between human IL-4 and c
c
.
Nevertheless, it will be important to understand how the c
c
mutations and the associated changes in IL-4 binding affect
the biological a ctivity of c
c
during IL-4 signaling or t he
signaling of the cytok ines that d epend on c
c
. Previous
experiments with IL-4 mutant proteins [15] revealed that
substitutions in the c

were aligned manually. The c entral part of the
EF1, BC2, and FG2 l oops of receptors are selectively shown. Key
residues fo r b inding are underlined. The d eletions are marked Ô–Õ.The
interaction of the receptor with the respective ligand is classified as
follows:
a
AD or AC helix interface involved in receptor binding;
b
polarity of the interface;
c
affinity of ligand–receptor interaction.
1496 J L. Zhang et al.(Eur. J. Biochem. 269) Ó FEBS 2002
mutant sequences derived from patients with XSCID
[58–61]. Two c
c
mutants (A134V and R202C) were found
to produce t wofold and fourfold reduced IL-4 and IL-2
binding, and to be less effective in modulating Jak3
activation stimulated by IL-4 and IL-2, respectively
[60,61]. A134 is located at the periphery of the c
c
epitope
identified in this study and has not been included in the
present experiment.
Some of the residues in the c
c
epitope for IL-4 binding as
identified in this study (Y103, L161, L208 and G210) have
been found to be mutated in patients with XSCID
(Fig 4B,C [62]). The XSCID phenotype seems to be caused

and hgp130 [51]. Our result and the mutagenesis analysis of
the b inding of the m urine c
c
chain t o I L-2 a nd IL-7 [30]
show that Y103 of c
c
is a key ligand-interacting residue for
IL-2, IL-4, and IL-7. Y103 is probably a common critical
residue for all c
c
-dependent receptor systems. In a ddition, in
that study [30], the counterpart of three dominated residues
I100, L 102 a nd L208 of human c
c
for I L-4 binding were
reported not to be important for IL-2 a nd IL-7 binding.
These residues are probably unique to IL-4 binding, a s
suggested by the fact that c
c
binding sites for different
cytokines overlap but are not identical [29,64]. However, it
cannot be ruled out that some of the binding residues of c
c
defined in our study also participate in IL-2 and IL-7
binding, as, in the aforementioned s tudy, only one re sidue
(Y103) was shown to be directly involved in IL-2 and IL-7
binding. Y 103A or Y103R mutations resulted in only
slightly (twofold to threefold) reduced IL-2 and IL-7
binding [30]. The difference between these results and our
own may partly originate f rom the different methods

globulin isotype switching. Curr. Opin. Immunol. 6, 838–846.
6. Shanafelt, A.B., Forte, C.P., Kasper, J.J., Sanchez-Pescador, L.,
Wetzel, M., Gundel, R. & Greve, J.M. (1998) An imm une cell-
selective interleukin 4 agonist. Proc. Natl Acad. Sci. USA 95,
9454–9458.
7. Srivannaboon, K., Shanafelt, A.B., T odisco, E., F orte, C.P.,
Behm, F.G., Raimondi, S.C., Pui, C.H. & Campana, D. (2001)
Interleukin-4 variant (BAY 36–1677) selectively induces apoptosis
in acute lymphoblastic leukemia cells. Blood 97, 752–758.
8. Sugamura, K., Asao, H., Kondo, M., T anaka, N., Ishii, N.,
Nakamura, M. & Takeshita, T. (1995) The common gam ma-chain
for multiple cytokine receptors. Adv. Immunol. 59, 225–277.
9. Gauchat, J.F., Schlagenhauf, E., Feng, N.P., Moser, R., Yamage,
M., Jeannin, P., Alouani, S., Elson, G ., Notarangelo, L.D., Wells,
T., Eugster, H.P. & Bonnefoy, J.Y. (1997) A novel 4-kb inter-
leukin-13 receptor alpha mRNA e xpressed in human B, T, and
endothelial cells encoding an alternate type-II interleukin-4/inter-
leukin-13 receptor. Eur. J . Immu nol . 27, 971–978.
10. Bazan, J.F. (1990) Structural design & molecular evolution of a
cytokine receptor superfamily. Proc. Natl A cad. Sc i. U SA 87 ,
6934–6938.
11. Wang, Y., Shen, B.J. & Sebald, W. (1997) A mixed-charge pair
in human interleukin 4 dominates high–affinity interaction
with the receptor alpha chain. Proc. Natl Acad. Sci. USA 94,
1657–1662.
12. Hage, T., Sebald, W. & Reinemer, P. (1999) Crystal structure of
the interleukin-4/receptor alpha chain com plex reveals a mo saic
binding interface. Cell 97, 271–281.
13. Zhang, J.L., Simeonowa, I., W ang, Y. & Sebald, W. (2002) The
high-affinity interaction of human IL-4 and the receptor a chain is

20. Takeshita, T., A sao, H., O htani, K., Ishii, N., K umaki, S.,
Tanaka, N., Munakata, H., Nakamura, M. & Sugamura, K.
(1992) Cloning of the gamma chain of the human IL-2 receptor.
Science 257, 379–382.
21. Russell, S.M., Keegan, A.D., Harada, N ., Nakamura, Y.,
Noguchi, M., Leland, P., Friedmann, M.C., Miyajima, A., Puri,
R.K., Paul, W.E. & Leonard, W.J. (1993) Interleukin-2 receptor
gamma chain: a functional co mponent of the in terleukin-4
receptor. Science 262 , 1880–1883.
22. Kondo, M., Takeshita, T., Hig uchi, M., Nakamura, M., S udo, T.,
Nishikawa, S. & Sugamura, K. (1994) Functional participation
of the IL-2 receptor g amma ch ain in IL -7 recep tor comp lexes.
Science 263, 1453–1454.
23. Leonard, W.J. & O’Shea, J.J. (1998) Jaks and STATs: biological
implications. Annu. Rev. Immunol. 16, 293–322.
24. Leonard, W.J. (1996) The molecular basis of X-linked severe
combined immunodeficiency: d efective cytokine receptor signal-
ing. Annu.Rev.Med.47, 229–239.
25. Lo, M., Bloom, M.L., Imada, K., Berg, M., Bollenbacher, J.M.,
Bloom, E.T., Kelsall, B.L. & Leonard, W.J. ( 1999) Restoration of
lymphoid populations in a murine m od el of X -lin ked severe
combined immun odeficien cy by a gene-therapy approac h. Blood
94, 3027–3036.
26. Soudais, C., Sh iho, T., Sharara, L.I., Guy-Grand, D., Taniguchi,
T., F ischer, A . & Di Santo, J.P. (200 0) Stable and f unctional
lymphoid re co nstitution of common cytokine receptor gamma
chain d eficient mice by r etrov iral-media ted gene transfe r. Blo od 95,
3071–3077.
27. Cavazzana-Calvo, M., Hacein-Bey, S ., de Saint Basile, G ., Gross,
F.,Yvon,E.,Nusbaum,P.,Selz,F.,Hue,C.,Certain,S.,Casa-

binding or receptor activation. EMBO J. 12, 5121–5129.
35. Schreiber, G. & Fersht, A.R. ( 1995) E nergetics of pr otein–protein
interactions: analysis of the barnase–barstar interface by
single mutations and do uble mutant cycles. J. Mol. Biol. 248,
478–486.
36. Kruse, N., Tony, H.P. & Sebald, W. (1992) Conversion of human
interleukin-4 into a high affin ity antagonist by a s ingle amino acid
replacement. EMB O J. 11, 3237–3244.
37. Sch reiber, G ., Frisch, C. & Fersht, A.R. (1997) The role of Glu73
of barnase in catalysis and t he binding o f b arstar. J. M ol. Biol. 270 ,
111–122.
38. Mueller, T., Oehlenschlaeger, F. & Buehner, M. (1995) Human
interleukin-4 and variant R88Q: phasing X-ray diffraction data
by molecular r eplacement using X-ray and nuclear magnetic
resonance models. J. Mol. Biol. 24 7, 360–372.
39. de Vos, A.M., U ltsch, M. & K ossiakoff, A .A. ( 1992) Human
growth hormone and extracellular domain of its recep tor: crystal
structure of the complex. Science 255, 306–312.
40. Bernstein, F.C., Koetzle, T.F., Williams, G.J.B., Meyer, E.F. Jr,,
Brice, M.D., Rodgers, J.R., K ennard, O., Shimanouchi, T. &
Tasumi, M. ( 1977) The P rotein Data Bank: a compute r-based
archival file for m a cromolecular structures. J. Mol. Bio l. 112,535–
542.
41. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991)
Improved meth ods f or b indin g prote in m od els i n e lectron density
maps an d the location of errors in these models. Act a Crystallogr.
A47, 110–119.
42. Bruenger, A.T. (1992) X-Plor, Version 3.1. A System for X-Ray
Crystallography and NMR. Yale University Press, New Haven,
CT, USA.

leads to the identification of c ontact s ites in th e i nter leukin-6
(IL-6). J. Biol. Chem. 272, 23748–23757.
51.Kurth,I.,Horsten,U.,Pflanz,S.,Dahmen,H.,Kuster,A.,
Grotzinger, J., Heinrich, P.C. & Muller-Newen, G. (1999) Acti-
vation of t he s ignal transducer glycoprotein 130 by both IL-6 and
IL-11 requires two dis tinct bind ing epitopes. J. Immunol. 162,
1480–1487.
52. Hoff man, R.C., Castner, B. J., Gerhart, M ., Gibson, M.G.,
Rasmussen,B.D.,March,C.J.,Weatherbee,J.,Tsang,M.,
Gustchina, A., Schalk-Hihi, C., Reshetnikova, L. & Wlodawer, A.
(1995) Direct evidence of a heterotrimeric complex of human
interleukin-4 with its receptors. Protein Sci. 4, 382–386.
53. Baker, D.P., Whitty, A., Zafari, M.R., Olson, D.L., Hession,
C.A., Miatkowski, K., Avedissian, L.S., Foley, S.F., McKay,
1498 J L. Zhang et al.(Eur. J. Biochem. 269) Ó FEBS 2002
M.L., Benjamin, C.D. & Burkly, L.C. (1998) The m urine a nti-
human co mmon gamma chain m onoclo nal a ntibody CP.B8
blocks the second step in the formation of the intermediate affinity
IL-2 receptor. Biochemistry 37, 14337–14349.
54. Hiraoka, O., Anaguchi, H., Asakura, A. & Ota, Y. (1995)
Requirement for the immunoglobulin-like domain of granulocyte
colony-stimulating factor receptor in formation of a 2: 1 receptor–
ligand complex. J. Biol. Chem. 270, 25928–25934.
55. Woodcock, J.M., Z acharakis, B., Plaetinck, G., Bagl ey, C.J.,
Qiyu, S., Hercus, T.R., Tavernier, J. & Lopez, A.F. (1994) Three
residues in the common beta c hain of the human GM -CSF, IL-3
and IL-5 receptors are essential for GM-CSF and IL-5 but not
IL-3 high affinity binding and in teract with Glu21 of GM-CSF.
EMBO J. 13, 5176–5185.
56. Wo odcock, J .M., B agley, C.J., Zacharakis, B. & L opez, A .F.

63. Malek, T.R., Porter, B.O. & He, Y.W. (1999) Multiple gamma
c-dependent cytokines r egulate T-cell develo pment. Immunol.
Today 20, 71–76.
64. He, Y.W., Adkins, B., Furse, R.K. & Malek, T.R. (1995)
Expression and function of the gamma c s ubunit of the IL-2, IL-4,
and IL-7 receptors. Distinct interaction of gamma c in the IL-4
receptor. J. Immunol. 154, 1596–1605.
Ó FEBS 2002 Mutagenesis of human c
c
ectodomain (Eur. J. Biochem. 269) 1499