Báo cáo khoa học: Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein potx

Amino acid residues on the surface of soybean 4-kDa peptide
involved in the interaction with its binding protein
Kazuki Hanada
1
, Yuji Nishiuchi
2
and Hisashi Hirano
1
1
Yokohama City University, Kihara Institute for Biological Research/Graduate School of Integrated Science, Yokohama, Japan;
2
Peptide Institute, Inc., Protein Research Foundation, Osaka, Japan
Soybean 4-kDa peptide, a hormone-like peptide, is a ligand
for the 43-kDa protein in legumes that functions as a protein
kinase and controls cell proliferation and differentiation. As
this peptide stimulates protein kinase activity, the interaction
between the 4-kDa peptide (leginsulin) and the 43-kDa
protein is considered important for signal transduction.
However, the mechanism of interaction between the 4-kDa
peptide and the 43-kDa protein is not clearly understood.
We therefore investigated the binding mechanism between
the 4-kDa peptide and the 43-kDa protein, by using gel-
filtration chromatography and dot-blot immunoanalysis,
and found that the 4-kDa peptide bound to the dimer form
of the 43-kDa protein. Surface plasmon resonance analysis
was then used to explore the interaction between the 4-kDa
peptide and the 43-kDa protein. To identify the residues of
the 4-kDa peptide involved in the interaction with the
43-kDa protein, alanine-scanning mutagenesis of the 4-kDa
peptide was performed. The 4-kDa peptide-expression
system in Escherichia coli, which has the ability to install

disulfide bonds into the target protein in the cytoplasm, was
employed to produce the 4-kDa peptide and its variants.
Using mass spectrometry, the expressed peptides were con-
firmed as the oxidized forms of the native peptide. Surface
plasmon resonance analysis showed that the C-terminal
hydrophobic area of the 4-kDa peptide plays an important
role in binding to the 43-kDa protein.
Keywords: hormone-like peptide; receptor-like protein;
protein–protein interaction; alanine-scanning mutagenesis;
surface plasmon resonance.
A 43-kDa protein in legume seeds has been shown to bind
to animal insulin [1]. This 43-kDa protein consists of
a (27 kDa) and b (16 kDa) subunits linked together with
disulfide bridge(s). The a-subunit has a cysteine-rich region
considered to be the interface for the interaction with its
ligand, and the b-subunit has protein kinase activity about
two-thirds that of the tyrosine kinase activity of rat insulin
receptor. Although proteins homologous to the 43-kDa
protein exist in different plant species [2–5], the biological
function of these proteins has not been completely clarified.
However, the 43-kDa protein from cotton has weak
antifungal activity against Alternaria brassicicola and Bot-
rytis cinerea [4]. As the 43-kDa protein is localized in plasma
membranes and cell walls [6], the 43-kDa protein is thought
to have receptor-like function.
This function, as a receptor-like protein, has allowed us
to assume the presence of a physiologically active ligand
which is capable of binding to the 43-kDa protein. A
4-kDa peptide was isolated from germinating soybean
seed radicles by affinity chromatography on a 43-kDa

protein-immobilized column [7]. The 4-kDa peptide is able
to stimulate protein kinase activity of the 43-kDa protein
[7]. The maximum stimulatory effect was observed at a
low concentration (1 n
M
) of the 4-kDa peptide, suggesting
that it is involved in signal transduction of the 43-kDa
protein [7]. The 4-kDa peptide is localized, in small
amounts, around the plasma membranes and cell walls
[7]. This subcellular localization is similar to that of the
43-kDa protein, suggesting that the 4-kDa peptide is
located at a site suitable for interaction with the 43-kDa
protein.
In a previous study we provided some evidence to show
that the 4-kDa peptide is physiologically active. The
4-kDa peptide was found to stimulate cell proliferation
and cell redifferentiation when added to the culture
medium of carrot callus tissue [8]. Furthermore, when
cDNA from the 4-kDa peptide was introduced into the
carrot callus, the transgenic callus grew rapidly compared
with the non-transgenic callus during the early stages of
development [8]. These results suggest that this peptide is
Correspondence to H. Hirano, Yokohama City University, Kihara
Institute for Biological Research/Graduate School of Integrated
Science, Maioka-cho 641-12, Totsuka, Yokohama, 244-8013 Japan.
Fax: + 81 45 820 1901; Tel.: + 81 45 820 1904;
E-mail:
Abbreviations: E. coli, Escherichia coli; IPTG, isopropyl thio-b-
D
-galactoside; PVDF, poly(vinylidene difluoride); SPR, surface

plasmon resonance; Trx, thioredoxin.
Enzymes: alkaline phosphatase (EC 3.1.3.1); lysylendopeptidase
(EC 3.4.21.50); restriction endonucleases EcoRI (EC 3.1.21.4)
and NcoI (EC 3.1.31.4); thioredoxin reductase (EC 1.8.1.9);
tyrosine kinase (EC 2.7.1.112).
Note: As the binding capabilities of insulin and the 4-kDa peptide to
the 43 kDa protein were similar, Watanabe et al. named the 4-kDa
peptide as leginsulin in their early publication. There are many con-
troversies related to the naming of this peptide as leginsulin. To avoid
confusion, in the present article we referred to the peptide as Ô4-kDa
peptideÕ instead of leginsulin.
(Received 28 February 2003, revised 16 April 2003,
accepted 22 April 2003)
Eur. J. Biochem. 270, 2583–2592 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03627.x
involved in the signal transduction mediated by the
43-kDa protein in carrot [8]. However, the molecular
mechanism of the interaction between the 4-kDa peptide
and 43-kDa protein is unknown. In previous work, we
determined the tertiary structure of the 4-kDa peptide by
NMR spectroscopy and found that this peptide belongs
to the T-knot superfamily [8]. The structure of the 4-kDa
peptide is similar to those of many growth factors in
animals, protease inhibitors and antimicrobial peptides in
plants, and toxins in insects [9]. As the function of these
molecules is to bind to their target proteins to regulate or
inhibit their activities, it is assumed that the function
of the 4-kDa peptide also relates to the regulation of the
43-kDa protein kinase activity.
In this work, we performed gel-filtration chromatography
to study the interaction between the 4-kDa peptide and the

43-kDa protein. We also investigated the binding mechan-
ism of the 4-kDa peptide, by alanine-scanning mutagenesis.
The results indicate that the hydrophobic region of this
peptide is important for binding to the 43-kDa protein. We
also describe the topological similarity of active residues
between the 4-kDa peptide and animal insulin.
Materials and methods
Materials
All oligonucleotides were obtained from Invitrogen Life
Technologies. The expression vector for Escherichia coli,
pET-32a[+], the expression host cell, BL21trxB (DE3),
and the BugBuster protein extraction reagent were
obtained from Novagen (Madison, WI, USA). A nickel-
chelating affinity chromatography column, HiTrap chelat-
ing HP (1 mL), and the gel-filtration chromatography
column for the SMART system, Superose 12 PC3.2/30,
were obtained from Amersham Bioscience (Uppsala,
Sweden). The size-standard proteins kit for gel-filtration
chromatography was purchased from Bio-Rad Laborat-
ories (Hercules, CA, USA). Biacore sensor chip CM5, was
obtained from Biacore (Uppsala, Sweden). The restriction
enzymes, EcoRI and NcoI, were from Nippon Gene
(Tokyo, Japan). All other inorganic and organic com-
pounds were purchased from WAKO Chemicals (Osaka,
Japan).
Gel-filtration chromatography
Gel-filtration chromatography was performed using the
SMART system in PC3.2/30 columns containing Superose
12 resin in 100 m
M

sodium phosphate/0.5
M
NaCl, pH 7.6.
The samples were eluted using the same buffer. Eighty
microlitres of fraction was collected in each tube subse-
quently, after discarding the exclusion volume. All gel
filtrations were carried out at room temperature. For gel
filtration using the Superose 12 resin, two sample solutions
were prepared. The first was the gel-filtration elution buffer
containing the 43-kDa protein incubated for 30 min at
room temperature; and the second was the gel-filtration
elution buffer containing a mixture of the 4-kDa peptide
and the 43-kDa protein [molecular concentration ratio:
2 : 1 (43-kDa protein : 4-kDa peptide)] incubated for
30 min at room temperature.
Dot-blot analysis
The eluted fractions of the gel filtration were spotted onto a
poly(vinylidene difluoride) (PVDF) membrane (10 lLper
spot). The membrane was blocked with 1% nonfat dry milk
in NaCl/Tris buffer (20 m
M
Tris/HCl, pH 7.4, containing
0.5
M
NaCl) for 1 h at room temperature. Polyclonal rabbit
anti-(4-kDa peptide) was dissolved in NaCl/Tris and
incubated with the membrane overnight at 4 °C. The
membrane was washed twice (10 min each wash) in NaCl/
Tris buffer at room temperature and incubated with goat
anti-(rabbit IgG) labeled with alkaline phosphatase. The

signal was detected with BCIP/NBT membrane phospha-
tase substrate (KPL, Gaithersburg, MD, USA).
Construction of the bacterial expression vector
and site-directed mutagenesis
The DNA sequence of the wild-type 4-kDa peptide was
amplified from the soybean 4-kDa peptide cDNA by PCR
using the following oligonucleotide primers: N-terminal
primer: 5¢-AAC CAT GGC TAA AGC AGA TTG TAA
TGGTGCATGT-3¢; C-terminal primer: 5¢-AAG AAT
TCTTATTATCCAGTTGGATGTATGCAGAA-3¢.
The amplified sequence was cloned into plasmid pET-
32a(+), via the NcoIandEcoRI restriction sites, into a
multicloning site located downstream of the S-Tag
sequence. This plasmid was termed pTrx-LEG. The validity
of the 4-kDa peptide DNA sequence was verified by
dideoxy sequencing. Site-directed mutagenesis was per-
formed, using pTrx-LEG as a template, according to the
methods of Higuchi et al. [10] and Ho et al.[11].All
residues of the 4-kDa peptide, with the exception of
alanines, cysteines, glycines and prolines, were singly
replaced by alanine. The resulting constructs were verified
by DNA sequencing. All of the mutational 4-kDa peptide
DNA sequences were recloned into the same restriction site
of the wild-type 4-kDa peptide DNA sequence.
Expression and purification of the 4-kDa peptide variants
E. coli BL21trxB(DE3) [F
–
ompT hsdS
B
(r

B
–
m
B
–
) gal dcm
trxB15::kan (DE3)], transformed with pTrx-LEG or the
corresponding variants, was grown at 37 °Cin1Lof
Luria–Bertani (LB) medium, containing 50 lg/mL carbeni-
cillin, until a D
600
value of 0.6 was reached. After addition of
isopropyl thio-b-
D
-galactoside (IPTG) to a final concentra-
tion of 1.0 m
M
, cells were grown for a further 4 h and
harvested by centrifugation at 6000 g for 10 min at 4 °C.
The cells were suspended in 40 mL of BugBuster protein-
extraction reagent. The cell suspension was incubated on
an orbital shaker, at a slow setting, for 10 min at room
temperature. In the soluble fraction, cell debris was removed
by centrifugation at 48 000 g for 15 min at 4 °C. The
supernatant was used as a crude extract. The Trx-tagged
4-kDa peptide, or its variants in the crude extract, were
purified according to immobilized metal affinity chroma-
tography. The crude extract was applied to HiTrap
chelating HP that immobilized Ni
2+

equilibrated with
20 m
M
sodium phosphate buffer (pH 7.4) containing 0.5
M
NaCl. The target protein was eluted with a 10–500 m
M
linear gradient of imidazole in 20 m
M
sodium phosphate
2584 K. Hanada et al. (Eur. J. Biochem. 270) Ó FEBS 2003
buffer (pH 7.4) containing 0.5
M
NaCl. The fractions
containing the target protein were combined.
Peptide mass fingerprinting
The Trx-tagged 4-kDa peptide, or its variants, were digested
with lysylendopeptidase (WAKO Chemicals). The digests
were desalted with ZipTip
l-C18
(Millipore, Boston, MA,
USA) and subjected to analysis by MALDI-TOF MS
(Tofspec 2E; Micromass, Manchester, UK). In MALDI-
TOF MS, ionization was accomplished with a 337-nm
pulsed nitrogen laser. Spectra were acquired in reflectron
using a 20-kV acceleration voltage. Samples were prepared
by mixing equal volumes of a 1–10 l
M
solution of the digests
and a saturated solution of a-cyano-4-hydroxycinnamic

acid as a matrix in 50% CH
3
CN with 0.1% trifluoroacetic
acid. Four microlitres of this mixture was spotted onto
thesampleplateandallowedtodesiccatetodryness.
The
MASSLYNX
software (Micromass) was used to analyze
the spectra.
Biacore
To confirm the mechanism of complex formation between
the 4-kDa peptide and the 43 kDa-protein, we employed
surface plasmon resonance (SPR) analysis using Biacore X
(Biacore). The purified wild-type 4-kDa peptide was
immobilized onto sensorchip CM5 according to the
supplier’s instructions. Different amounts of 43-kDa pro-
tein, dissolved in running buffer (20 m
M
sodium phosphate,
pH 7.4, containing 0.5
M
NaCl), were injected as analytes
for binding analysis at 25 °C using a flow rate of
20 lLÆmin
)1
.
The binding affinities of the Trx-tagged wild-type
4-kDa peptide and its variants were determined using
Biacore X, to measure the association rate constant (k
a

)
Fig. 1. Gel-filtration chromatography of the
43-kDa protein and the 4-kDa peptide/43-kDa
protein complex. (A) Chromatogram of the
43-kDa protein. (B) Chromatogram of the
4-kDa peptide and the 43-kDa protein com-
plex. The elution points of the size-standard
proteins are shown with arrows: BGG, bovine
gamma globulin (158 kDa); OA, ovalbumin
(44 kDa); MG, equine-myoglobin (17 kDa);
VB12, vitamin B
12
(1.35 kDa). Lines have
been used in each chromatogram to separate
the fractions. (C) Dot-blot analysis of the
fraction shown in panel B. Fractions, as given
in B were spotted onto a poly(vinylidene
difluoride) membrane. The fractions contain-
ing the 4-kDa peptide were detected using
anti-(4-kDa peptide). Numbers refer to the
fractions shown in panel B. Underlined
numbers indicate the presence of the 4-kDa
peptide. See the Materials and methods for
further details.
Ó FEBS 2003 Interaction between soybean peptide and its binding protein (Eur. J. Biochem. 270) 2585
and the dissociation rate constant (k
d
). The 43-kDa
protein was immobilized onto sensorchip CM5, according
to the supplier’s instructions, to yield approximately 5560

response units of covalently coupled protein. Kinetic
analysis was carried out by injecting three serial dilutions
(400 n
M
,800n
M
and 1.6 l
M
) of Trx-tagged 4-kDa
peptide or variants in running buffer (20 m
M
sodium
phosphate, pH 7.4, containing 0.5
M
NaCl)at25°Cusing
a flow rate of 20 lLÆmin
)1
.
Fitting sensorgram data was carried out according to
global fitting, and the k
a
and k
d
values were calculated with
a 1 : 1 Langmuir model using the
BIAEVALUATION
software,
version 3.2 RC2 (Biacore). The dissociation constant (K
D
)

was calculated as K
D
¼ k
d
/k
a
.
Results and discussion
Identification of a complex of 4-kDa peptide
and 43-kDa protein
We first sought to determine the potential association of the
43-kDa protein, as the receptor of the physiologically active
peptide usually forms an oligomer to activate the function of
the receptor [12]. When the 43-kDa protein was subjected
to gel-filtration chromatography, we observed only one
peak for a complex of 80-kDa, suggesting that the 43-kDa
protein is present as a dimer (Fig. 1A). Subsequently, we
applied the solution containing the 43-kDa protein and
4-kDa peptide to the gel filtration column, and observed a
peak with almost the same retention time as that of the
80-kDa complex. We studied proteins containing these
fractions by dot-blot analysis using anti-(4-kDa peptide).
The result revealed that both the 4-kDa peptide and 43 kDa
protein were present in the same fractions, suggesting that
the 4-kDa peptide interacts with the dimer of 43-kDa
protein.
To determine the K
d
of the 4-kDa peptide and 43-kDa
protein, the wild-type 4-kDa peptide was immobilized onto

sensorchip CM5 by amine coupling. The 43-kDa protein
solution was passed through the flow cells as an analyte.
Interaction of ligand and analyte was detected in real time as
a change in the SPR signal. The association and dissociation
sensorgrams obtained are shown in Fig. 2. The K
d
of the
4-kDa peptide for binding to the 43-kDa protein was
calculated as 1.86 · 10
)8
M
.
Interaction of Trx-tagged 4-kDa peptide
with the 43-kDa protein
The Trx-tagged 4-kDa peptide was expressed in a
thioredoxin-reductase gene (TrxB) null mutant,
BL21trxB(DE3), and purified according to immobilized
metal affinity chromatography (Fig. 3). The binding activity
of the 4-kDa peptide to the 43-kDa protein is dependent on
the maintenance of its tertiary structure by three intra-
molecular disulfide bonds. The reduced 4-kDa peptide has
significantly less activity than the oxidized form of the
Fig. 2. Representative surface plasmon resonance sensorgrams of
binding between the 43-kDa protein and the 4-kDa peptide were
dependent on concentration. Details of the procedure are described in
the Materials and methods. Phases before the asterisk (*) represent the
association sensorgrams; phases after the asterisk represent the disso-
ciation sensorgrams. The kinetic parameters, association rate constant
(k
a

) and dissociation rate constant (k
d
), were calculated using
B
IAEVALUATION
software; k
a
¼ 5.28 · 10
4
M
)1
Æs
)1
and k
d
¼ 9.85 ·
10
)4
Æs
)1
. The dissociation constant, K
D
, was calculated as K
D
¼ k
a
/k
d
;
K

D
¼ 1.86 · 10
)8
M
.
Fig. 3. Coomassie blue-stained SDS/PAGE
gels showing the Trx-tagged 4-kDa peptide
variants. The number of each lane corresponds
to the position which introduced variation:
panel A D2A–D19A; and panel B R21A–
T36A. Lanes M, the molecular marker; lane T,
Trx-tag; and lane W, Trx-tagged wild-type
4-kDa peptide. Arrows show Trx-tag and
Trx-tagged 4-kDa peptide variants. (C) Sites
of mutations induced in the 4-kDa peptide.
The sites are shown by open boxes. Ser17 was
not substituted to alanine because the side-
chain was buried inside the 4-kDa peptide.
2586 K. Hanada et al. (Eur. J. Biochem. 270) Ó FEBS 2003
peptide [7]. To introduce the intramolecular disulfide bonds
in the expressed 4-kDa peptide, we used BL21trxB(DE3)
host cell, TrxB null mutant and pET-32a[+] vector.
Bessette et al. [13] described that this strain can form
Fig. 4. MALDI-TOF MS analysis of wild-
type 4-kDa peptide. (A) Mass spectrum of the
wild-type 4-kDa peptide. Trx-tagged wild-type
4-kDa peptide was digested with lysylendop-
eptidase and subjected to MALDI-TOF MS.
The 4-kDa peptide was observed as 3S–S
form, 3916.70 m/z ([M+H]

+
), marked by
circling. (B) Theoretical mass of each oxidized
form of the 4-kDa peptide. The column of
oxidized form shows the number of intra-
molecular disulfide bonds (S–S).
Table 1. Identification of the oxdized form of 4-kDa peptide variants by
MALDI-TOF MS. 3S–S denotes the formation of three intramolec-
ular disulfide bonds.
Trx-tagged variant
Theoretical mass
[M+H]
+
(m/z)
Observed mass
[M+H]
+
(m/z)
WT (3S–S) 3916.72 3916.70
D2A (3S–S) 3872.73 3872.64
N4A (3S–S) 3873.71 3874.01
S8A (3S–S) 3900.72 3900.54
F10A (3S–S) 3840.79 3840.62
E11A (3S–S) 3858.71 3858.64
V12A (3S–S) 3888.69 3888.56
R16A (3S–S) 3831.65 3831.17
R18A (3S–S) 3831.65 3831.20
D19A (3S–S) 3872.73 3872.61
R21A (3S–S) 3831.65 3831.37
V23A (3S–S) 3888.69 3888.21

I25A (3S–S) 3874.67 3874.57
L27A (3S–S) 3874.67 3874.51
F28A (3S–S) 3840.79 3841.07
V29A (3S–S) 3888.69 3889.06
F31A (3S–S) 3840.79 3841.02
I33A (3S–S) 3874.67 3874.50
H34A (3S–S) 3850.70 3851.22
T36A (3S–S) 3886.71 3886.58
Fig. 5. Representative sensorgrams of binding between analytes (Trx-
tagged wild-type 4-kDa peptide and Trx-tag) and ligand (43-kDa pro-
tein). (A) Binding of Trx-tagged wild-type 4-kDa peptide. (B) Binding
of Trx-tag. Phases before the asterisk (*) represent the association
sensorgrams; phases after the asterisk represent the dissociation
sensorgrams.
Ó FEBS 2003 Interaction between soybean peptide and its binding protein (Eur. J. Biochem. 270) 2587
disulfide bonds more efficiently in the cytoplasm than in the
oxidizing environment of the periplasmic space. Stewart
et al. [14] showed that Trx, which serves as an oxidant
instead of a reductant, mediates disulfide bond formation in
the thioredoxin-reductase null mutant because the reduction
system in the cytoplasm does not work. By peptide mass
fingerprinting, we confirmed that the expressed 4-kDa
peptide has three intramolecular disulfide bonds (Fig. 4,
Table 1). This result indicates that we can construct various
alanine substitution-variants crosslinked with disulfide
bonds using this expression system.
The purified 43-kDa protein was immobilized onto
sensorchip CM5 and confirmed to bind to the Trx-tagged
4-kDa peptide by SPR analysis. The K
d

of the Trx-tagged
4-kDa peptide for the 43-kDa protein was determined as
8.56 · 10
)8
M
(Fig. 5A, Table 2). It should be noted that
the K
d
value reported here is higher than that previously
described for the wild-type 4-kDa peptide, probably because
of changes in the source of the 4-kDa peptide (see the
Materials and methods for further details). To investigate
whether Trx-tag impedes binding of the 4-kDa peptide to
the 43-kDa protein, Trx-tag expressed in E. coli transformed
with pET-32a[+] was injected to the 43-kDa protein-
coupling sensorchip. In this experiment, we did not observe
any sensorgrams showing that Trx-tag bound to the 43-kDa
protein (Fig. 5B). This result shows that the 4-kDa peptide
and 43-kDa protein, but not Trx-tag, are involved in
binding of the Trx-tagged 4-kDa peptide to the 43-kDa
protein.
The 4-kDa peptide in the expressed Trx-tagged 4-kDa
peptide has three intramolecular disulfide bonds. As it had a
binding activity similar to that of the wild-type 4-kDa
peptide, we concluded that the intramolecular disulfide
bonds were correctly formed in the Trx-tagged 4-kDa
peptide.
Dissociation constants of the 4-kDa peptide variants
To investigate the residues of the 4-kDa peptide involved in
binding to the 43-kDa protein, we generated 4-kDa peptide

variants, in which 19 residues were substituted with alanine
using pTrx-LEG as a template. To avoid potential struc-
tural perturbation, alanine, cysteine, glycine and proline
residues were not substituted. All variants were generated as
Trx-tagged proteins and purified according to the methods
used for the wild-type 4-kDa peptide. The purity of the
fused proteins was confirmed on a Coomassie blue-stained
SDS/PAGE gel. All purified proteins were detected as major
bands with the expected molecular weights (Fig. 3). The
number of disulfide bonds in the variants was investigated
by peptide mass fingerprinting, and all variants were found
to have three disulfide bonds (Table 1). The K
d
values for
binding to the 43-kDa protein were investigated by SPR
analysis, as employed for the wild-type 4-kDa peptide.
The results of our analyses of the 4-kDa peptide alanine
variants are shown in Table 2 and Fig. 6. Figure 6 shows
the ratio of the K
d
value of the 4-kDa peptide variant to the
K
d
value of the wild-type 4-kDa peptide. Of the 19 alanine
Table 2. Association rate constants (k
a
), dissociation rate constants (k
d
) and dissociation constants (K
D

) for binding alanine variants of Trx-tagged
4-kDa peptide to 43-kDa protein. Dissociation constants were calculated as follows: K
D
¼ k
d
/k
a
.RelativeK
D
values were calculated as: K
d
variants/
K
d
wild type.
Trx-tagged variant k
a
(10
3
M
)1
Æs
)1
) k
d
(10
)4
M
)1
Æs

)1
) K
D
(10
)8
M
) Relative K
D
Leginsulin WT 5.63 4.82 8.50 1.00
A. Charged to alanine variants
D2A 3.05 15.20 49.80 5.81
E11A 4.35 2.05 4.71 0.550
R16A 9.38 6.24 6.65 0.777
R18A 7.41 3.61 48.70 5.69
D19A 8.74 6.28 7.18 0.839
R21A 6.99 6.36 9.09 1.06
B. Aromatic to alanine variants
F10A 12.20 3.47 2.85 0.333
F28A 0.36 12.00 333.00 38.90
F31A 1.10 102.00 927.00 108.00
C. Polar to alanine variants
N4A 1.44 14.30 99.00 11.60
S8A 2.01 13.80 68.70 8.02
H34A 3.02 19.20 63.60 7.43
T36A 4.73 38.20 80.80 9.44
D. Fatty to alanine variants
V12A 1.13 11.00 97.30 11.40
V23A 6.03 9.30 15.40 1.80
I25A 2.32 53.50 231.00 27.00
L27A 3.38 12.30 36.20 4.23

V29A 1.30 129.00 994.00 116.00
I33A 1.60 62.70 392.00 45.80
2588 K. Hanada et al. (Eur. J. Biochem. 270) Ó FEBS 2003
variants, 13 caused a significant impairment in binding of
the 43-kDa protein, i.e. greater than a fourfold increase in
the K
d
value. Three of the 13 variants (Asp2, Asn4 and Ser8)
are located in the N-terminus of the 4-kDa peptide and their
K
d
values for the 43-kDa protein increase from five- to
12-fold. Two variants, Val12 and Arg18, which showed a
six- and 11-fold increase in K
d
, respectively, are located in
the loop between the first and the second strand in the
4-kDa peptide. His34 and Thr36 variants, located in the
C-terminus of the 4-kDa peptide, result in a seven- and
ninefold increase in K
d
, respectively. The other variants
(Ile25, Leu27, Phe28, Val29, Phe31, Ile33), whose residues
constitute the hairpin-b motif, caused a remarkable decrease
in affinity for the 43-kDa protein, ranging from fourfold
(Leu27) to 116-fold (Val29). These variants were classified
into several groups, and it was found that hydrophobic and
aromatic residues contributed remarkably to the increase
of K
d

for the 43-kDa protein (Table 2); in particular, five
residues (Ile25, Phe28, Val29, Phe31 and Ile33) play a
critical role in binding to the 43-kDa protein.
Role of amino acids in the 4-kDa peptide
By alanine-scanning mutagenesis of the 4-kDa peptide, we
identified that 13 amino acids play an important role in the
interaction between this peptide and the 43-kDa protein.
Eleven amino acids among the 13 mutants were organized
into two discontinuous fragments (fragment 1 and fragment
2). Fragment 1 comprised the N-terminal region (Asp2–
Ser8), while fragment 2 constituted the C-terminal region
(Ile25–Thr36) (Fig. 6C). As the mutations of fragment 2
result in a higher increase in K
d
than those of fragment 1,
fragment 2 was considered to play a more important role in
affinity for the 43-kDa protein. Of the 11 amino acids, one is
charged, four are polar, four are hydrophobic and two are
aromatic. The higher number obtained of aromatic and
hydrophobic residues emphasized the importance of these
amino acids in the interaction between the 4-kDa peptide
and the 43-kDa protein. The secondary structures of these
two fragments, as revealed from NMR spectroscopy of the
4-kDa peptide, indicate that fragment 1 contains the loop
Fig. 6. Structure of the functional epitopes of the 4-kDa peptide. The Ca backbone of the 4-kDa peptide is shown as a tube representation (A, B and
C). The mutated amino acids are shown in space-filling representation. Alanine variants of amino acids, shown in white, had no effect on affinity.
Those in yellow produced a two- to 10-fold reduction in affinity, and those in orange had a 10- to 100-fold reduction in affinity. Alanine variants of
amino acids, shown in red, had a >100-fold decrease in affinity. (D) Summary of alanine scanning of the 4-kDa peptide. The results are expressed as
the ratio of dissociation of the variant to that of the wild-type. The amino acids mutated to alanine are designated by a single-letter code.
Ó FEBS 2003 Interaction between soybean peptide and its binding protein (Eur. J. Biochem. 270) 2589

and b-strand, and fragment 2 contains hairpin-b [14]. These
structures form the sheet of the putative binding area
(Fig. 7A,B). Of the two fragments, fragment 2 appears to be
the most important in binding to the 43-kDa protein.
Mutation of Val29 and Phe31 to alanine resulted in the
43-kDa protein with the lowest affinity, and substitution of
Ile25 and Ile33 with alanine produced a 20-fold higher K
d
than found in the wild-type protein (Table 2). Interestingly,
all of the residues in fragment 2 were located at the same
region, forming a hydrophobic patch (Figs 6 and 7A,B,C).
The other residues, charged or polar, of fragment 2
surrounded this hydrophobic patch. The residues of frag-
ment 1 were also found in the surrounding hydrophobic
patch (Figs 6 and 7A,B,C). These topological alignments
suggest that the hydrophobic residues, Val29 and Phe31,
play a central role in binding to the 43-kDa protein and that
the wall consisting of fragment 1 and part of fragment 2
contributes to binding of the 4-kDa peptide to the 43-kDa
protein (Fig. 7A,B,C).
In Fig. 6C, we identified that two amino acids (Val12 and
Arg18), in addition to the 11 residues described above, were
involved in binding to the 43-kDa protein. The substitution
of Val12 and Arg18 to alanine affected binding to the
43-kDa protein. Unexpectedly, the side-chains of these two
residues were oriented in a different direction from those of
fragment 1 and fragment 2, which indicates that Val12 and
Arg18 do not belong to fragment 1 and fragment 2 and
indicates that Val12 and Arg18 might play a different role
from those residues of fragment 1 and fragment 2. Further

analysis of the interaction between the 4-kDa peptide and
43-kDa protein is required.
Several reports suggest that the decreases in affinity
observed in these types of mutations directly effect
receptor–ligand interaction, rather than misfolding, of
variant proteins [15]. Alanine substitution is reported to
be nondisruptive for globular protein structure [16]. In the
4-kDa peptide, three intramolecular disulfide bonds are
important for maintaining the tertiary structure. In the
present study, peptide mass fingerprinting showed that,
similarly to the wild-type 4-kDa peptide, all alanine
variants possessed three disulfide bonds (Fig. 4, Table 1).
Furthermore, all variants have a k
a
value which is
similar to that of wild-type peptide, suggesting that
substitution with alanine has no effect on the tertiary
Fig. 7. The location of fragment 1 and fragment 2 in the 4-kDa peptide tertiary structure, and comparison of the tertiary structure of insulin and the
4-kDa peptide. (A and B) Fragment 1 and fragment 2 are shown in orange and red, respectively. (C) Hydrophobic potential surfaces of the 4-kDa
peptide. According to hydrophobicity, the molecular surface is colored on a gradient from red (negative hydrophobicity) to blue, passing through
white at a hydrophobicity of zero. (D) Inactive state of insulin (1ai0). (E) Active state of insulin (1hit). (F) The 4-kDa peptide (1ju8). The residues
that constitute the insulin receptor-binding area are shown in red, and the residues important for the direction to insulin receptor are shown in
orange (D and E). In (F), four residues in red are most influenced by alanine substitution, and two residues in orange are located in a similar space as
the residues in orange of (E). The opened yellow squares showed a similar topology of side-chains of putative active residues in both insulin and the
4-kDa peptide (E and F).
2590 K. Hanada et al. (Eur. J. Biochem. 270) Ó FEBS 2003
structure of the 4-kDa peptide (Table 2). Exceptionally,
mutation of Phe28 to alanine caused a decrease in k
a
(Table 2), as it is located in the loop of the hairpin-b

motif and this area is also exposed to the solvent. This
suggests that the aromatic residue, Phe28, plays a vital
role in maintaining the hairpin-b during interaction with
solvent.
Interaction of insulin with the 43-kDa protein
Similarly to the 4-kDa peptide, insulin is able to interact
with the 43-kDa protein [1]. If the 4-kDa peptide and
insulin share the same manner of binding to the 43-kDa
protein, topological similarity of critical residues should
exist in the two peptides, as the two peptides do not share
the same fold. We have hypothesized previously that the
area consisting of Val23, Val29, Phe31 and Ile33 in the
4-kDa peptide [8] is involved in binding to the 43-kDa
protein because of topochemical similarity to the active
area of insulin consisting of ValA3, TyrA19, ValB12 and
TyrB16 (Fig. 7D,E,F). In the active state, insulin exposes
the active area (ValA3, TyrA19, ValB12 and TyrB16) for
entry into the insulin receptor (Fig. 7E) [17]. Among the
mutations of these four residues in the 4-kDa peptide
(Val23, Val29, Phe31 and Ile33), three (Val29, Phe31 and
Ile33) were involved in affinity for the 43-kDa protein.
Instead of Val23, Ile25 was found to be important for
binding to the 43-kDa protein. The topology of the side-
chains of Ile25, Val29, Phe31 and Ile33 in the 4-kDa
peptide was similar to that of the active area in insulin
(Fig. 7F). If the mechanism of the interaction between the
4-kDa peptide and 43-kDa protein has the minimum
components of insulin–insulin receptor interaction, the area
consisting of Ile25, Val29, Phe31 and Ile33 in the 4-kDa
peptide should play a critical role in the interaction with

the 43-kDa protein. These results suggest that there might
exist, on the surface of the 43-kDa protein, an area that
consists of hydrophobic residues facing the hydrophobic
patch in the 4-kDa peptide.
On the other hand, the C-terminal b-strand area, PheB25
and TyrB26, in insulin is required to direct the insulin
receptor [18–22]. When the 4-kDa peptide was compared to
the active state of insulin, Leu27 and Phe28 of the 4-kDa
peptide could occupy a similar place as PheB25 and TyrB26
of insulin (Fig. 7E,F). Therefore, it is suggested that Leu27
and Phe28 share the same role as PheB25 and TyrB26 in
insulin.
The area consisting of Ile25, Val29, Phe31 and Ile33 in the
4-kDa peptide is important for interaction with the 43-kDa
protein (Figs 6 and 7D,E,F). Although Leu27 and Phe28
are also involved in the interaction with 43-kDa protein, the
role of these residues is probably different from that of the
four residues (Ile25, Val29, Phe31 and Ile33). Generally,
the hydrophobic triplet of PheB24, PheB25 and TyrB26 of
the C-terminal B-chain domain of insulin is important for
directing the affinity of insulin receptor interaction [18–22].
As Leu27 and Phe28 of the 4-kDa peptide are located in the
same region against the aromatic triplet, Leu27 and Phe28
in the 4-kDa peptide probably regulate the orientation of
interaction with the 43-kDa protein.
Although the 43-kDa protein is not identical to the insulin
receptor, they show a resemblance in some structural
architecture. For example, both proteins form a dimer,
while their protomers consist of two disulfide-linked a and b
subunits, contain a cysteine-rich region in their a subunits,

and show protein kinase activity in their b subunits. As
mentioned above, the interaction system between 4-kDa
peptide and 43-kDa protein may be similar to the insulin–
insulin receptor interaction system.
Acknowledgements
We thank Prof. F. X. Avile
´
s and Dr N. Islam for their invaluable
suggestions during this work. We also thank Dr M. Takaoka for her
help in producing the recombinant 4-kDa peptide. This work was
supported in parts by grants for the National Project on Protein
Structural and Functional Analysis to H.H.
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