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

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

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

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

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

)
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

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

) 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

+
), 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

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

) 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

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

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

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

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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