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: hirano@yokohama-cu.ac.jp 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. References 1. Komatsu, S., Koshio, O. & Hirano, H. (1994) Protein kinase activity and insulin-binding activity in plant basic 7S globulin. Biosci. Biotechnol. Biochem. 58, 1705–1706. 2. Satoh, S., Sturm, A., Fujii, T. & Chrispeels, M.J. (1992) cDNA cloning of an extracellular dermal glycoprotein of carrot and its expression in response to wounding. Planta 188, 432–438. 3. Kolivas, S. & Gayler, K.R. (1993) Structure of the cDNA coding for conglutin c, a sulphur-rich protein from Lupinus angustifolius. Plant Mol. Biol. 21, 397–401. 4. Chung, R.P T., Neumann, G.M. & Polya, G.M. (1997) Puri- fication and characterization of basic proteins with in vitro anti- fungal activity from seed of cotton, Gossypium hirsutum. Plant Sci. 127, 1–16. 5. Poltronieri, P., Cappello, M.S., Dohmae, N., Conti, A., Fortu- nato, D., Pastorello, E.A., Ortolani, C. & Zacheo, G. (2002) Identification and characterisation of the IgE-binding proteins 2S albumin and conglutin gamma in almond (Prunus dulcis)seeds. Int. Arch. Allergy Immunol. 128, 97–104. 6. Nishizawa, N.K., Mori, S., Watanabe, Y. & Hirano, H. (1994) Ultrastructural localization of the basic 7S globulin in soybean (Glycine max) cotyledons. Plant Cell Physiol. 35, 1079–1085. 7. Watanabe, Y., Barbashov, S.F., Komatsu, S., Hemmings, A.M., Miyagi, M., Tsunasawa, S. & Hirano, H. (1994) A peptide that stimulates phosphorylation of the plant insulin-binding protein: isolation, primary structure and cDNA cloning. Eur. J. Biochem. 224, 167–172. 8. Yamazaki, M., Takaoka, M., Katoh, E., Hanada, K., Sakita, M., Sakata, K., Nishiuchi, Y. & Hirano, H. (2003) A possible phy- siological function and the tertiary structure of a 4-kDa peptide in legumes. Eur. J. Biochem. 270, 1269–1276. 9. Lin, S.L. & Nussinov, R. (1995) A disulphide-reinforced structural scaffold shared by small proteins with diverse functions. Nat. Struct. Biol. 2, 835–837. 10. Higuchi, R., Krummel, B. & Saiki, R.K. (1988) A general method of in vitro preparation and specific mutagenesis of DNA frag- ments: study of protein and DNA interactions. Nucl. Acids Res. 16, 7351–7367. 11. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59. 12. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. & Watson, J.D. (2002) Molecular Biology of the Cell, 4th edn. Garland Pub- lishing, New York. 13. Bessette, P.H., A ˚ slund,F.,Beckwith,J.&Georgiou,G.(1999) Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc. Natl. Acad. Sci. USA 96, 13703–13708. Ó FEBS 2003 Interaction between soybean peptide and its binding protein (Eur. J. Biochem. 270) 2591 14. Stewart, E.J., A ˚ slund, F. & Beckwith, J. (1998) Disulfide bond formationintheEscherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. EMBO J. 17, 5543–5550. 15. Mariuzza, R.A., Phillips, S.E. & Poljak, R.J. (1987) The structural basis of antigen–antibody recognition. Annu. Rev. Biophys. Bio- phys. Chem. 16, 139–159. 16. Wells, J.A. (1991) Systematic mutational analysis of protein– protein interfaces. Methods Enzymol. 202, 390–411. 17. Pittman, I., IV, Nakagawa, S.H., Tager, H.S. & Steiner, D.F. (1997) Maintenance of the B-chain beta-turn in [GlyB24] insulin mutants: a steady-state fluorescence anisotropy study. Biochem- istry 36, 3430–3437. 18. Nakagawa, S.H. & Tager, H.S. (1986) Role of the phenylalanine B25 side chain in directing insulin interaction with its receptor: steric and conformational effects. J. Biol. Chem. 261, 7332–7341. 19. Nakagawa, S.H. & Tager, H.S. (1987) Role of the COOH- terminal B-chain domain in insulin–receptor interactions: identification of perturbations involving the insulin mainchain. J. Biol. Chem. 262, 12054–12058. 20. Mirmira, R.G., Nakagawa, S.H. & Tager, H.S. (1991) Importance of the character and configuration of residues B24, B25, and B26 in insulin–receptor interactions. J. Biol. Chem. 266, 1428–1436. 21. Mirmira, R.G. & Tager, H.S. (1989) Role of the phenylalanine B24 side chain in directing insulin interaction with its receptor: importance of main chain conformation. J. Biol. Chem. 264, 6349–6354. 22. Mirmira, R.G. & Tager, H.S. (1991) Disposition of the phenyl- alanine B25 side chain during insulin–receptor and insulin–insulin interactions. Biochemistry 30, 8222–8229. 2592 K. Hanada et al. (Eur. J. Biochem. 270) Ó FEBS 2003 . Amino acid residues on the surface of soybean 4-kDa peptide involved in the interaction with its binding protein Kazuki Hanada 1 ,. 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