Báo cáo Y học: Functional epitope of common c chain for interleukin-4 binding

Functional epitope of common cchain 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 act on target cells through an IL-4

receptor complex consisting of the IL-4 receptor achain and

the common cchain (c

). An IL-4 epitope for c

binding has

previously been identified. In this study, the c

residues

involved in IL-4 binding were defined by alanine-scanning

mutational analysis. The epitope comprises c

residues I100,

L102, and Y103 on loop EF1 together with L208 on loop

FG2 as the major binding determinants. These predomin-

antly hydrophobic determinants interact with the hydro-

phobic IL-4 epitope composed of residues I11, N15, and

Y124. Double-mutant cycle analysis revealed co-operative

interaction between c

and IL-4 side chains. Several c

residues involved in IL-4 binding have been previously

shown to be mutated in X-linked severe combined

immunodeficiency. The importance of these binding residues

for c

function is discussed. These results provide a basis for

elucidating the molecular recognition mechanism in the IL-4

receptor system and a paradigm for other c

-dependent

cytokine receptor systems.

Keywords:commoncchain; interleukin 4; mutagenesis;

protein–protein interaction; structure/function.

Interleukin-4 (IL-4) is a multifunctional cytokine that plays

a critical role in the regulation of immune responses [1,2]. It

induces the generation of Th2-dominated early immune

response [3] and determines the immunoglobulin class

switching to IgE [4]. Dysregulation of IL-4 function is

strongly correlated with type I hypersensitivity reactions,

such as allergies and asthma [5]. The IL-4 receptor complex

is therefore a potential target for the development of

antiallergic drugs. The central role of IL-4 in the develop-

ment of Th2 cells suggests that it may be of benefit in the

treatment of autoimmune disease characterized by an

imbalance of Th cells [6]. Its ability to induce growth arrest

and apoptosis in leukemic lymphoblasts in vitro [7] suggests

that IL-4 is also a promising cytokine for the treatment of

high-risk acute lymphoblastic leukemia. Understanding the

molecular recognition mechanism in the IL-4 receptor

system is a prerequisite for the rational design of IL-4-like

drugs.

IL-4 is one of the short-chain four-helix bundle cytokines.

Its effects depend on binding to and signaling through a

receptor complex consisting of a primary high-affinity

binding subunit, the IL-4Ra, and a low-affinity receptor,

depending on the cell type, the common cchain (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, which is characterized by the presence

of at least one cytokine-binding homology region (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]. The fibronectin type III domain is

comprised of seven bstrands, the sequences of which are

conserved between members of the family, while loop

sequences connecting the bstrands vary between family

members and putatively contain residues that mediate

distinct intermolecular contacts. These loop regions were

therefore selected for this mutational analysis.

A comprehensive mutational analysis of IL-4 in which

single residues were replaced by alanine or charged residues

yielded high-resolution data on the binding epitopes for 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 located on helices

A, B, and C [11]. The importance of site 1-binding

determinants and their partner residues on IL-4Ra(D72,

Y183 as key binding determinants) was subsequently

confirmed and further defined by determining the crystal

structure of the 1 : 1 IL-4/IL-4Raectodomain (IL-4-

binding protein, IL-4BP) complex [12] and by mutational

analysis of the IL-4BP binding epitope [13]. The results have

already been used for the rational design of IL-4 minipro-

teins [14]. The IL-4 site 2 epitope for c

comprises residues

I11 and N15 on helix A together with Y124 on helix D as

major binding determinants and three minor determinants

K12, R121, and S125 on helices A and D [15]. A double

mutant of IL-4 that completely inhibits responses induced

by IL-4 and IL-13 by disrupting the binding of the IL-4 site

2 epitope to c

or IL-13Ra1provedtobeaverypromising

anti-asthma drug [16–18]. Two further IL-4 mutants that

selectively inhibit IL-4-induced activity on endothelial cells

appeared to be good candidate drugs for the treatment of

certain autoimmune diseases [6] and high-risk acute

lymphoblastic leukemia [7]. However, the residues on c

that contribute to IL-4 site 2 binding remain uncertain.

Correspondence to W. Sebald, Theodor-Boveri-Institut fu

¨rBiowis-

senschaften (Biozentrum), Physiologische Chemie II, Universita

¨t

¨rzburg, Am Hubland, D-97074 Wu

¨rzburg, Germany.

Fax: + 49 931 888 4113, Tel.: + 49 931 888 4111,

E-mail: sebald@biozentrum.uni-wuerzburg.de

Abbreviations: IL-4, interleukin-4; IL-4Ra, interleukin-4 receptor a

chain; IL-4BP, IL-4 binding protein; c

, common cchain; 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, human

erythropoietin receptor; b

,commonbchain.

(Received 14 November 2001, revised 16 January 2002, accepted

21 January 2002)

Eur. J. Biochem. 269, 1490–1499 (2002) ÓFEBS 2002

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, IL-21 [19]. c

alone binds ligands with very

low affinity (K

150 l

for IL-4) [15]. Recruitment of c

into receptor complexes for the above cytokines increases

receptor affinity for binding [20–22]. c

participates in

cytokine signaling in several receptor complexes via JAK3

[23]. Mutations of either c

or JAK3 result in X-linked

severe combined immunodeficiency (XSCID) which is

characterized by a failure in T and NK cell development

[24]. c

-knockout mice have been generated and their

immune system successfully reconstituted by gene therapy

[25,26]. Initial attempts at gene therapy for patients with

XSCID had been successful for more than 10 months

[27,28]. Thus, defining the IL-4-binding determinants on c

is important not only for elucidating molecular recognition

and activation mechanisms in the IL-4 receptor system and

possibly providing a paradigm for other c

-dependent

cytokine receptor systems, but also for delineating the

molecular pathology of XSCID.

So far, the binding epitopes of human and murine c

for some c

-dependent cytokines have been studied.

A molecular mapping study using the antagonistic

monoclonal antibody PC.B8, which reacts with a discon-

tinuous site on human c

, localized c

binding residues to

four loops, but did not identify single specific residues for

ligand binding [29]. Mutational analysis of murine c

employing heterodimeric IL-2R and IL-7R on whole cells

suggests that c

epitopes for IL-2 and IL-7 binding

overlap and comprise at least three distinct putative loop

segments of the c

protein [30]. Here we report the effect

of single amino-acid substitutions in the human c

ectodomain on IL-4 binding. Biosensor techniques

employing soluble recombinant IL-4, IL-4-BP and the

wild type or mutant forms of human c

ectodomain

revealed the contributions of c

residues to IL-4 binding.

The possible co-operativity between some residues on the

epitope and the IL-4 site 2 epitope was analyzed by

double-mutant cycle analysis.

EXPERIMENTAL PROCEDURES

Protein expression and purification

The ectodomain of human c

comprising amino-acid

residues 1–232 [20] was expressed with a C-terminal

thrombin cleavage site (LVPRGS) plus a His

tag in SF9

insect cells according to the manufacturer’s instructions

(PharMingen). The protein was isolated from the culture

medium of infected SF9 cells by standard procedures

involving Ni

/nitriloacetate/agarose (Qiagen), digested

with thrombin (Sigma), and purified by gel-filtration

chromatography through a Superdex 200 HR 10/30 col-

umn (Pharmacia). After exhaustive dialysis against water,

the purified protein was freeze-dried and stored at )80 °C.

ThecDNAforthemurinec

ectodomain comprising

residues 1–233 [31] was cloned into the temperature-

regulated expression vector pRpr9 fd [32], expressed in

Escherichia coli strain KS 474, and refolded as described

[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 A182, 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 purified to homogeneity as described [11,34]. Protein

concentrations were determined by measuring A

280

,using

an absorption coefficient (e

280

)¼8860

Æcm

for

IL-4, e

280

¼7370

Æcm

for A124 IL-4, e

280

66 930

Æcm

for IL-4BP, e

280

¼61 450

Æcm

for

human c

280

¼60 170

Æcm

for A103 human c

and e

280

¼45 660

Æcm

for murine c

Mutagenesis of the c

ectodomain

cDNA for human c

ectodomain was submitted to in vitro

cassette mutagenesis employing synthetic double-stranded

oligonucleotides. The c

variants were expressed and

purified as the wild-type human c

ectodomain.

Biosensor interaction analysis

The binding of c

variants to IL-4/IL-4BP was recorded on a

BIAcore 2000 system (Pharmacia Biosensor) 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 com-

mand COINJECT: (a) IL-4 at 0.1 l

in HBS buffer (10 m

Hepes, pH 7.4, 150 m

NaCl, 3.4 m

EDTA, 0.005%

surfactant P20) was perfused over flow cells 1 and 2 at a flow

rate of 10 lLÆmin

at 25 °C for 2 min; (b) 0.1 l

IL-4 plus

ectodomain or c

variants at 1–10 l

inthesamebuffer

were perfused in the same way for 2 min; (c) HBS buffer

alone was perfused for 5 min; (d) free receptors were

regenerated by perfusion with 0.1

acetic acid/1

NaCl

for 30 s. Sensograms were recorded at a data-sampling rate

of 2.5 Hz and evaluated as described [15]. Equilibrium

binding of c

variants at 1, 2, 3, 5, 10 l

was measured for at

least three times in duplicate. The mean standard deviation

(mean r) was 13.8% ± 6.5% for the K

values calculated

from the five variant concentrations. For the double mutant

cycle analysis [35], the same procedure as above was used

except that IL-4 variants [15,36] at 0.1 l

and c

variants at

2, 4, 6, 10, 20 l

were perfused (the mean rwas

16.4% ± 7.4% for the K

values). The loss of binding free

energy on mutation for IL-4 and c

wascalculatedasddG

(kJÆmol

)¼5.69 log K

(mutant)/K

(wild-type). The

interaction energy between two residues was calculated by

the double-mutant cycle method as in Eqn. (1):

ddGint ¼ddGX-AþddGY-BÿddGX-A;Y-Bð1Þ

where ddG

X-A

and ddG

Y-B

are the changes in binding

energy on mutation of X to A and Y to B (mutation of IL-4

and c

in this experiment), respectively, and ddG

X-A, Y-B

the

change on the simultaneous mutation of X to A and Y to B.

ddG

int

is a measure of the co-operativity of the interaction

of the two components mutated. ddG

int

¼0indicatesthat

the pair of residues analyzed do not 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,a¼0.95) calculated

from the mean for ddG

int

areshowninTable3.

ÓFEBS 2002 Mutagenesis of human c

ectodomain (Eur. J. Biochem. 269) 1491

Molecular modeling of the IL-4–IL-4BP–c

ternary

complex

The present model is based on the crystal structure of the

complexofIL-4andIL-4BP(PDBentry1IAR[12]),

augmented by the model of c

derived from human growth

hormone receptor (hGHR), as obtained from an older

model (T. Mueller, & W. Kammer, personal communica-

tion, Universita

¨tWu

¨rzburg, Germany)

1of the ternary

complex of IL-4–IL-4BP–c

. This old model was based on

the structure of free IL-4 (PDB entry 1HIK [38]) and of

models of the extracellular domains of IL-4Raand c

obtained by analogy modeling following the structure of the

hGHR complex (PDB entry 3HHR [39]). The 3HHR data

were obtained from the protein databank (PDB [40]). The

old model was built in such a way that all cysteine residues

formed proper disulfide bonds, and all evidence from

mutation experiments available at the time was used to

adjust the binding epitopes of the receptor chains. The

resulting alignment required some nontrivial rebuilding with

insertions and deletions, and, consequently, the resulting

model of the IL-4 receptor complex had to be extensively

energy refined. The program

[41] was used for model

building, and the program

PLOR

[42] for energy refinement.

The differences between the experimentally determined

binary complex and the corresponding components of the

old model were significant in detail, but the gross changes

were small enough that the binding topology of c

could be

transferred to the new model without major problems.

The local program

DISDM

2 was used (H. J. Hecht, &

M. Buehner, unpublished results) to build and adjust the

present model using the data of mutational analysis of IL-4

and 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 program

EREF

was used [43], which is called from within

DISDM

RESULTS

Site-specific mutagenesis of amino acids

in the c

ectodomain

Alanine substitutions were targeted to residues in four

putative interconnecting loops and the interdomain segment

of the human c

ectodomain based on the published models

[44–46], and sequence alignment performed between c

and

several cytokine receptors, the major ligand-binding deter-

minants of which were identified. These include the hGHR

[39,47], the human erythropoietin receptor (hEPOR

[48,49]), IL-4BP [12], and the human gp130 (hgp130

[50,51]). Eighteen c

variants were generated with amino-

acid substitutions in the AB1, EF1, BC2, FG2 loops and the

interdomain segment (Fig. 1). A deletion mutant lacking

residues 1–33 of the N-terminus of c

,namedc

CHR, was

also generated to find out whether this N-terminal region of

is required for ligand binding.

All human c

wild-type or variant proteins could be

purified to apparent homogeneity by Ni

/nitrilotriacetate/

agarose and gel filtration. The wild-type human c

ectodo-

main expressed in SF9 cells was recovered as monomeric

and dimeric species [52,53]. The murine c

ectodomain

expressed in E. coli occurred as a monomer (Fig. 2). Initial

biosensor studies showed that the different forms of human

and murine proteins exhibited similar binding affinity for

the IL-4–IL-4BP complex. The mixture of monomeric and

dimeric human c

interacts with the complex with a K

Fig. 1. Amino-acid substitutions in the ectodomain of the human com-

mon cchain (c

). The amino-acid sequence of c

is shown with boxed

portions indicating predicted b-strands which are designated by the

letter below the box. Residues substituted in this study are indicated by

asterisks.

Fig. 2. Gel-filtration analysis of the human c

ectodomain expressed in

SF9 cells and the murine c

ectodomain expressed in E. coli. The samples

were applied to a Superdex 200 HR 10/30 column and eluted with the

same buffer. The two peaks of human c

represent dimer (A) and

monomer (B).

1492 J.-L. Zhang et al.(Eur. J. Biochem. 269)ÓFEBS 2002

, and murine c

with a K

of 1.6 l

(Fig. 3 and

Table 1). In addition, different preparations of wild-type

human c

ectodomain consistently showed a K

of 4 l

irrespective of the monomer to dimer ratio (data not

shown). Therefore, the mixtures of dimeric and monomeric

human c

protein were used for all biosensor measurements.

The c

epitope for IL-4 binding

The method of measuring the binding of c

to IL-4–IL-4BP

by biosensor was established previously [15]. The dissocia-

tion constant K

evaluated from the concentration

dependence of equilibrium binding proved to be very

reliable for measuring the interaction of the c

ectodomain

with IL-4–IL-4BP. The measured K

for interaction of c

ectodomain variants with IL-4–IL-4BP are compiled in

Table 1. Eight c

variants including c

CHR exhibited

unchanged binding characteristics. Changes in binding

affinity were observed in 11 c

variants. The K

of six

variants was too high to be reliably determined. A rough

estimate yields K

values of about 200–300 l

for I100A,

L102A, Y103A and L208A, and K

values of about 500–

1000 l

for C160A and C209A. The K

values of five

variants, N128A, H159A, L161A, E162A, and G210A,

were found to be increased threefold to fourfold compared

with the K

of wild-type c

, suggesting that these residues are

part of the c

binding interface, but do not play a key role in

binding. The loss of binding affinity of the four variants

I100A, L102A, Y103A and L208A is not likely to be caused

by extended structural alterations, as I100A, L102A, and

L208AwerereportedtobindtoIL-2andIL-7withthe

same affinity as wild-type c

, and the Y103A mutation

resulted in only twofold to threefold reduced IL-2 and IL-7

binding [30]. Thus, the four residues I100, L102, Y103 and

L208 are hot spots on c

, contributing > 9 kJÆmol

each.

The five minor residues investigated contribute only 2.9–

3.5 kJÆmol

. The two cysteine variants C160A and C209A

exhibited a largely reduced binding affinity (K

> 500 l

Thismaybecausedbystructuralperturbationofthe

protein. A direct role in binding, however, cannot be

excluded for these residues.

Double-mutant cycle analysis of the IL-4–c

interface

The co-operativity of the interaction of some residues on the

IL-4 site 2 epitope and the c

ectodomain was determined in

this experiment. Double-mutant cycles were constructed

only for the mutants with minimal effects on binding

(Tables 2 and 3), because the K

values for the interaction

between variants of the main binding residues I100, L102,

Y103, and L208 of c

andIL-4variantsaswellasthe

interaction between variant I11A of IL-4 and c

variants

were too high to be reliably determined. Interaction between

residues on IL-4 and c

can be grouped according to their

coupling energies (Table 3). The main binding determinant

of IL-4 Y124 failed to exhibit positive coupling energies with

any of the c

residues analyzed. IL-4 Y124 probably

interacts with the main functional side chains of the receptor

located on loop EF1 (I100, L102, Y103), the binding of the

alanine variants of which was too weak to be analyzed by

this approach. Remarkably, IL-4 S125 neighboring Y124

does show coupling to receptor N128 in addition to that to

Fig. 3. Sensograms recording the binding of human and murine c

ectodomains to the IL-4–IL-4BP complex. IL-4BP was immobilized on

the biosensor matrix. At time zero, perfusion with 100 n

IL-4 was

initiated. The saturation binding of IL-4 was arbitrarily set as zero.

After 120 s, perfusion was continued with 100 n

IL-4 plus c

ecto-

domain. In different cycles, 5 l

human (a) or murine (b) c

ecto-

domains were applied. Perfusion with buffer alone started at time

240 s. The ruler indicates resonance units (RU) corresponding to

1–10 l

murine c

ectodomain. The resonance unit for 5 l

human c

corresponded to that for 1.6–2 l

murine c

Table 1. Equilibrium binding between c

ectodomain mutants and

IL-4–IL-4BP. The dissociation constants K

were evaluated from

equilibrium binding between wild-type (wt) or mutants (mut) of the c

ectodomain and immobilized IL-4BP saturated with IL-4. The loss of

free energy of binding on mutation was calculated as ddG

(kJÆmol

)¼5.69 log K

(mut)/K

(wt).

Alanine

variant

Equilibrium binding

ddG

(kJÆmol

)

(mut)/K

(wt)

Murine c

(wt) 1.6

Human c

(wt) 4.0 1.0 0.0

Human c

CHR 4.5 1.1 0.2

Loop 1 (AB1)

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

ÓFEBS 2002 Mutagenesis of human c

ectodomain (Eur. J. Biochem. 269) 1493

G210. The IL-4 side chain of N15 functionally interacts with

the central receptor side chain N128, and also with H159

located at the periphery of the functional c

epitope. The

relative positions of the coupling side chains as proposed by

our theoretical model of the ternary complex (see below) are

presented in the open-book view in Fig. 4A,B. The two

receptor side chains N1128 and H159 are 12 A

˚apart in the

model. This could indicate that our c

model is

inaccurate, because this model does not completely fit the

results of the double-mutant cycle analysis. Alternatively,

the interaction of IL-4 side chain N15 with H159 (coupling

energy only 0.8 kJÆmol

) may be indirect. Of particular

interest is the IL-4 side chain of R121, which, after being

substituted with D or E, leads to a selective IL-4 agonist

specifically impaired in IL-13Ra1 binding [6,7,16,17,34].

The IL-4 R121 was distinct in showing positive coupling

during interaction with the c

side chain L161.

Model of the structure of the IL-4–IL-4BP–c

ternary

complex

In a series of steps, c

was adapted to achieve a good fit to

the core structure (the binary complex; Fig. 5). The

procedure started 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 part of the model, but some minor

changes in side chain orientation could not be avoided for

proper adaptation. An important point was to keep the

C-terminal domains of the receptor chains close together, as

this was expected to be essential for dimer formation and

thereby signaling through the membrane. The structures of

and the ternary complex were modeled so that residues

that exhibit positive coupling energies during double-

mutant cycle analysis were placed close to each other.

Occasionally, however, there was a Ôconflict of interestÕ

between the requirements of interaction and those of

dimerization.

DISCUSSION

This mutational analysis defines human c

residues involved

in IL-4 binding. The residues are located in the EF1, BC2,

and FG2 loops and the interdomain segment of c

.The

functional binding epitope of c

includes residues I100,

L102, Y103, and L208 as major binding determinants and

five residues, N128, H159, L161, E162, and G210, as minor

determinants. Our results also show that the truncated c

CHR has the same binding affinity as the complete c

ectodomain, indicating that the short N-terminal region of

is not required for ligand binding. This is true for most

type I cytokine receptors, except for hgp130 [51] and

granulocyte colony-stimulating factor receptor [54]. There-

fore, c

CHR, the short form of the c

ectodomain, may be

Table 2. Double mutant cycle analysis of interaction between c

and

IL-4. The dissociation constants K

were evaluated from equilibrium

binding between wild-type (wt) or mutants (mut) of the c

ectodomain

and immobilized IL-4BP saturated with wild-type or mutants of IL-4.

The loss of free energy of binding on mutation was calculated as

ddG ¼5.69 log K

(mut)/K

(wt). ddG

sum

is the sum of the losses of free

energy of binding upon mutation for IL-4 and c

separately. ND,

Sensogram could not be evaluated because of weak binding.

IL-4

variants

chain

variants

)

ddG

(kJÆmol

)

ddG

sum

(kJÆmol

)

wt wt 4.0

N15A wt 20 4.0

R121A wt 12 2.8

Y124F wt 6.9 1.4

S125A wt 8.5 1.9

wt N128A 15 3.2

wt H159A 17 3.5

wt L161A 13 2.9

wt E162A 13 2.9

wt G210A 15 3.2

N15A N128A 34 5.3 7.2

N15A H159A 61 6.7 7.5

N15A L161A 75 7.2 6.9

N15A E162A ND – 6.9

N15A G210A 59 6.7 7.2

R121A N128A 50 6.2 6.0

R121A H159A 75 7.2 6.3

R121A L161A 27 4.7 5.7

R121A E162A 127 8.6 5.7

R121A G210A 81 7.4 6.0

Y124F N128A 22 4.2 4.6

Y124F H159A 33 5.2 4.9

Y124F L161A 24 4.5 4.3

Y124F E162A 83 7.5 4.3

Y124F G210A 29 4.9 4.6

S125A N128A 19 3.9 5.1

S125A H159A 57 6.6 5.4

S125A L161A 53 6.4 4.8

S125A E162A 37 5.5 4.8

S125A G210A 22 4.2 5.1

Table 3. Co-operativity between residue pairs in the interaction interface of c

and IL-4. The coupling energy between a pair of residues was calculated

as ddG

int

¼ddG

sum

)ddG (data from Table 2) according to eqn (1). The underlined values indicate favorable interaction. The numbers in

parentheses are the calculated errors (2 r,a¼0.95). ND, Sensogram could not be evaluated because of weak binding.

chain

variants

ddG of IL-4 variants (kJÆmol

)

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)ÓFEBS 2002

Báo cáo Y học: Functional epitope of common c chain for interleukin-4 binding

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