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The binding of copper ions to glycine-rich proteins (GRPs) from Cicer arietinum
Biochimica et Biophysica Acta 1722 (2005) 69 – 76 http://www.elsevier.com/locate/bba
Regular paper
The binding of copper ions to glycine-rich proteins (GRPs) from Cicer arietinum Masakatsu Kamiyaa,b, Yasuhiro Kumakia, Katsutoshi Nittaa, Takeshi Matsumotob, Kunio Hikichib, Norio Matsushimab,* a
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan b School of Health Sciences, Sapporo Medical University, Sapporo 060-8556, Japan Received 21 August 2004; received in revised form 10 November 2004; accepted 11 November 2004 Available online 10 December 2004
Abstract Cicer arietinum GRP1 and GRP2 are rich in glycine interposed with histidine and tyrosine. In order to study whether or not these proteins bind Cu2+, circular dichroism (CD) and nuclear magnetic resonance (NMR) were measured for three synthetic peptides corresponding to sections of the protein’s sequences including 1, N1Y2G3H4G5G6G7N8Y9G10N11, where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH2 group at the C-terminus. The visible CD spectra for 1 showed a positive peak near 590 nm not at pH 6.0 but pH 7.4 in the presence of copper ions. The Cu2+ binding induced a drastic change in the far-UV CD spectra, showing the occurrence of large conformation changes. In the 2D TOCSY NMR spectra at pH 7.4, the addition of small amounts of CuSO4 caused a significant broadening of proton resonances of not only His4 but also Gly5, Asn8 and Asn11. CD titration experiment suggested that NYGHGGGNYGN including one repeat unit comprises the fundamental Cu2+ binding unit. D 2004 Elsevier B.V. All rights reserved. Keywords: Glycine-rich protein; Copper binding; Histidine/glycine/tyrosine-rich domain; CD; NMR
1. Introduction GRP1 and GRP2 proteins from Cicer arietinum belong to the family of glycine-rich proteins (GRPs) [1]. mRNAs encoding GRP1 and GRP2 accumulate in chickpea plants in response to fungal infection and other stress factors such as wounding and drought treatment [1]. However, the structure and function of GRP1 and GRP2 are still unknown. GRPs have been implicated in structural cell wall components like hydroxyproline-rich glycoproteins (HRGPs) and prolinerich proteins (PRPs) [1–3]. GRP1, with 148 amino acids (PIR accession no. T09527), and the GRP2 fragment, presumably lacking a part of the N-terminal domain (EMBL accession no. AJ01289), show the same basic pattern: an N-terminal region with structural similarities to putative cell wall signal * Corresponding author. Tel.: +81 11 611 2111; fax: +81 11 612 3617. E-mail address: [email protected] (N. Matsushima). 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.11.012
peptides of GRPs from other plants followed by a region of glycine-rich repeats (Fig. 1). The glycine-rich region is rich in glycine interposed with histidine and tyrosine (HGY-rich domain). The HGY-rich domain contains tandem repeats of heptapeptide GGGNYG(H/N) (Fig. 1). The heptapeptide repeats 10 in GRP1, whereas GRP2 has 17 repeats. The HGY/W-rich domains are present in the ozoneinducible proteins OI2-2 and OI14-3 from Atriplex canescens, and mammalian prions that are responsible for the transmissible spongiform encephalopathies [4], as seen in C. arietinum GRPs. The HGY/W-rich domains in ozone-inducible proteins and prions contain tandem repeats of hexapeptide YGHGGG and octapeptide PHGGGWGQ, respectively. These HGY/W-rich domains have shown to bind copper ions [5–10]. However, the Cu2+-binding unit differs from each other. One copper ion is bound to each octapeptide repeat in prions [6,7], while likely two hexapeptide repeats occur in ozone-inducible proteins [10].
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80Ts column (Tosoh Coop. Japan). Finally, all peptides were chemically blocked with an acetyl group (Ac) at the Nterminus and an -NH2 group at the C-terminus. Blocking the N-terminus is important since polypeptide N-terminal amines can bind directly to copper ions. 2.2. Circular dichroism (CD)
Fig. 1. Schematic diagram of the amino acid sequence of glycine-rich proteins GRP1 and GRP2 from Cicer arietinum (A) and the tandem repeats of hexapeptide GGGNYG(H/N) (B). The signal peptide is hatched, and the glycine-rich domain, which is largely different from the consensus sequence, is dotted.
All CD spectra were recorded on a JASCO J-725 spectropolarimeter (Jasco, Japan). The peptide solutions were concentrated to 0.1 mM (for Far-UV CD) and 0.4 mM (for visible CD) in 10 mM phosphate buffer at 25 8C. Two pHs were prepared: pH 6.0 and 7.4. Individual peptide solutions were prepared in the absence of Cu2+ and presence of Cu2+ with a molar ratio of 1:1. Typically, a cell with a 1.0-mm pathlength was used to obtain spectra between 190 and 260 nm with sampling points every 0.2 nm. The average of the five points was plotted. A 1-cm pathlength cell was used for data between 300 nm and 800 nm, also with 0.5-nm sampling intervals. The average of the four points was plotted. Direct CD measurements (h, in mdeg) were converted to molar ellipticity [h] using the relationship [h]=h/cln in 190–260 nm and h/cl in 300–800 nm, where c is the peptide concentration, l is the path length and n is the number of peptide bonds in the sequence. Molar ellipticity [h] is expressed in units deg cm2 dmol 1. 2.3. NMR spectroscopy
This leads to the hypothesis that the repetitive HGY-rich domains in C. arietinum GRPs may also bind copper ions. If so, what the segment of the Cu2+-binding unit is? In order to verify this, circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements were performed using synthetic peptides corresponding to sections of the sequences of GRPs. The present results demonstrate that N1Y2G3H4G5G6G7N8Y9G10N11 binds one Cu2+.
All NMR spectra were measured on JEOL ALPHA 500 or 600 spectrometers. Sequence-specific assignments of
2. Materials and methods 2.1. Peptide samples Considering the experimental observations on copper binding to the synthetic peptides (including YGHGGGY and HGGGY) corresponding to sections of the sequences of ozone-inducible proteins [10], three peptides for experimental studies on Cu2+-binding were designed. These three synthesized peptides correspond to sections of the protein’s sequences that are truncated and/or frame-shifted analogues of the basic repeating units: 1 (NYGHGGGNYGN), 2 (HGGGNY) and 3 (HGGGNYGN). The three peptides were purchased from SIGMA Genosys Japan K.K. (Ishikari, Japan). All peptides were chemically synthesized using solid-phase Fmoc chemistry on an Applied Biosystems Japan Ltd Pioneer peptide synthesizer. Synthetic peptides were purified by reverse-phase HPLC using a TSKgel ODS-
Fig. 2. Far-UV CD (190–260 nm) of peptide 1 (Ac-NYGHGGGNYGNNH2). Peptide concentrations are 0.1 mM in 10 mM phosphate buffer at pH 7.4, 25 8C. The figure shows the CD spectra with the addition of Cu2+ in 0.0 (o), 0.2 (5), 0.4 (w), 0.6 (), 0.8 (+) and 1.0 ( ) mole-equivalents of CuSO4.
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proton resonances for only peptide 1 were obtained by 2D TOCSY (mixing time=80 ms) and 2D ROESY (mixing time=250 ms) experiments [11,12]. The 2D NMR spectra were measured in the phase-sensitive mode using TPPIState for quadrature detection in f1. The water signal was suppressed by the DANTE method in TOCSY and ROESY [12]. Peptide concentrations were 3.5 mM in H2O/D2O (90%/10%), 10 mM phosphate buffer and 0.5 mM NaN3, pH 6.0 at 10 8C and 2.1 mM in pH 7.4. The spectra of 2D TOCSY (mixing time=80 ms) at pH 7.4 were also measured with the addition of Cu2+ in 0.008, 0.034 and 0.064 moleequivalents of CuSO4. The 1D 1H-NMR spectra was performed in 2.5 mM in H2O/D2O (90%/10%), pH. 7.4, 10 mM phosphate buffer and 0.5 mM NaN3 at 10 8C. CuSO4 was added in aliquots of 0.002 mole-equivalents up to 0.034 mole-equivalents. DSS was added to the solutions as an internal reference (0.0 ppm).
3. Results 3.1. Far-UV CD spectra The far-UV CD spectra for peptide 1 were compared at pH 6.0 and pH 7.4 in the absence and the presence of Cu2+ ions. In the absence of Cu2+, the spectrum was unaffected by pH (data not shown). However, the addition of Cu2+ revealed a remarkable, pH-dependent structural change (Fig. 2). A large conformational change was observed in the presence of Cu2+ at pH 7.4; the CD spectrum was dominated by two, new positive peaks near 207 nm and 226 nm in which the latter has been attributed to aromatic groups of tyrosyl residues [6,13,14] (Fig. 2). These two peaks are comparable to positive peaks at 200 nm and 229 nm observed for YGHGGGY in the ozone-inducible proteins, respectively [10]. The large, drastic change in the
Fig. 3. Visible CD spectrum (300–800 nm) of the three peptides, 1 (AcNYGHGGGNYGN-NH2) (A), 2 (Ac-HGGGNY-NH2) (B) and 3 (AcHGGGNYGN-NH2) (C). Peptide concentrations are 0.4 mM in 10 mM phosphate buffer at 25 8C. o, the CD spectra with the addition of Cu2+ in 1.0 mole-equivalents of CuSO4 at pH 7.4; , the CD spectra under the same conditions at pH 6.0.
.
Fig. 4. Direct Cu2+ binding curves for peptide 1 (Ac-NYGHGGGNYGNNH2). Change in [h] with Cu2+ was at 592 nm.
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CD spectra by the addition of Cu2+ indicates that peptide 1 binds Cu2+ at pH 7.4. 3.2. Visible CD spectra When Cu2+ is added to the peptides, CD bands arising due to d–d transitions appear in the visible region. These transitions become observable by CD only when Cu2+ is in a chiral ligand environment, and thus trace amounts of aqueous Cu2+ ions are not detected. The CD spectra for the three peptides, 1, 2 and 3, at pH 6.0 and pH 7.4 are shown in Fig. 3. The binding of Cu2+ to the three
peptides exhibited pH-sensitivity. At pH 6.0, CD bands arising due to d–d transitions were not observed regardless of the presence of Cu2+ (Fig. 3). In contrast, the characteristic CD bands were observed in the presence of Cu2 at pH 7.4. For peptide 1, the CD spectrum had a positive peak near 590 nm with a molecular ellipticity equal to +3.15103, while in the CD spectra peptide 2 and 3 appeared to show very weak peak near 590 nm. The ellipticity for 1 (NYGHGGGNYGN) was very comparable to that of YGHGGGY, YGHGGGYGHGGGY, PHGGGWGQ and HGGGW [6,8,10]. Two other peaks were also observed: a
Fig. 5. Fingerprint region and the ChH region of the 2D-TOCSY spectra and fingerprint regions of the 2D-ROESY spectra of peptide 1 (Ac-N1-Y2-G3-H4-G5G6-G7-N8-Y9-G10-N11-NH2). The 2D-TOCSY spectra is in the upper figure and the 2D-ROESY spectra is in the lower figure. The spectra at pH 6.0 and pH 7.4 are shown in black and red, respectively.
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positive peak near 320 nm due to charge transfer and a negative peak near 480 nm (Fig. 3), while PHGGGWGQ and HGGGW showed a positive peak at 340 nm and a negative band near 720 nm [6,9]. The visible CD spectra observed here clearly indicate that peptide 1 at least binds Cu2+. 3.3. CD titration The CD signals were obtained as a function of titrated Cu2+ to determine the stoichiometry of metal ion to the peptide binding (Fig. 4). The positive CD signal intensity was followed as a function of added Cu2+ at pH 7.4. The titration curve for peptide 1 is shown in Fig. 4. The curve appears to show curvature in the region of about 1.0 added equivalent. The binding curves may be influenced by conformational change dependent on the copper concentration.
resonances linked to the paramaganetic centers by through-bond (contact) or through-space (pseudocontact) interactions. The low levels of Cu2+ in the 1H NMR study enable the observation of significant differential broadening of the resonances of protons close to the copper binding site, as long as the exchange rate of the copper is rapid. The effects of Cu2+ on the 1H NMR spectrum of peptide 1 were studied to determine the nature of the copper-binding sites. The first step of this study was the identification of individual amino acid residues and the sequential assignment in the absence of Cu2+ ions. The identification and assignments were completed unambiguously by the TOCSY and ROESY experiments at pH 6.0 (Fig. 5A,B). Next, the proton assignment at pH 7.4 was performed using the following two procedures: (1)
3.4. NMR assignments (2) The slow electron spin relaxation of the paramagnetic Cu2+ results in marked broadening of proton NMR
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The identification and assignments at pH 7.4 were completed by the comparison the TOCSY spectra at pH 6.0 and pH 7.4 (Fig. 5A). The 1D spectrum at pH 7.4 was compared with the 2D spectrum at pH 7.4 and the 1D and 2D spectra at pHs 6.0 and 7.4. The spectral assignment of the five
Fig. 6. The 2D-TOCSY spectra showing the effects of the addition of Cu2+ to peptide 1 (Ac-N1-Y2-G3-H4-G5-G6-G7-N8-Y9-G10-N11-NH2) (2.1 mM in 90%/10% H20/D2O, pH 7.4, and 10 mM phosphate buffer at 10 8C. The spectra in the absence of Cu2+ are shown in black while the spectra with the addition of Cu2+ in 0.064 mole-equivalent of CuSO4 are shown in red. In A–C, squared cross peaks are observed in the absence of Cu2+.
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glycine residues was performed using a fitting program (http://nakamura-2.ees.hokudai.ac.jp/NakamuraX/nmr/ nmr.html).
In the 1D spectra at pH 7.4, the side chain protons (Cy2H, C H, and CaH) of His4 were unambiguously assigned (Fig. 7A,B). The backbone CaH protons of the five glycine E1
Fig. 7. 1H NMR plots showing the effects of the addition of Cu2+ to peptide 1 (Ac-N1-Y2-G3-H4-G5-G6-G7-N8-Y9-G10-N11-NH2) (2.5 mM in 90%/10% H2O/D2O, pH 7.4, and 10 mM phosphate buffer at 10 8C. (A) 6.5–8.0 ppm; (B) 2.5–3.3 ppm; (C) 3.7–4.1 ppm. The upper, middle and bottom of the figure shows the spectra in the absence of Cu2+ and with the addition of Cu2+ in 0.008 and 0.034 mole-equivalents of CuSO4, respectively.
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residues (Gly3, Gly5, Gly6, Gly7 and Gly10) were also reasonably assigned (Fig. 7C). 3.5. Cu2+ complexation studies of peptide 1 by 1H NMR The addition of small amounts of CuSO4 to 1 caused differential broadening of resonances, which indicated the location of copper binding. The effects of Cu2+ addition on the 2D-TOCSY and 1D spectra of 1 are shown in Figs. 6 and 7. There was a general increase in the linewidth of most resonances with increasing Cu2+ concentration. After the addition of 0.064 mole-equivalents of Cu2+, the resonances of the backbone CaH protons of His4, Gly5 and Asn8, and the ChH protons of Asn8 and Asn11 were so broad that they were indistinguishable from the baselines, whereas other resonances remained relatively sharp (Fig. 6A–C). The significant broadening of the His4 CaH resonance was observed even after the addition of 0.034 mole-equivalent. After this addition, the side chain protons (ChH, CE1H and Cy2H) of His4 were also broadened significantly more than other resonances (Figs. 7A,B). The Gly5 CaH resonance was broadened more strongly than those of the other glycine residues (Fig. 7C), which is consistent with the differential broadening observed in the 2D-TOCSY spectra (Fig. 6B). Moreover, the CE1H and Cy2H protons of His4 in the presence of Cu2+ were downfield from those in the absence of Cu2+. Such downfield shifts are consistent with the observed change in the far-UV CD spectra, indicating structural change induced by the binding of Cu2+ to 1.
Fig. 8. Plausible structure for the complex of Cu2+ with peptide 1 (Ac-N1Y2-G3-H4-G5-G6-G7-N8-Y9-G10-N11-NH2).
ion (Fig. 3). Thus, it is suggested that the NYGHGGGNYGN segment comprises the Cu2+-binding unit. This binding unit predicts that GRP1 and GRP2 bind at least four and eight copper ions per protein, respectively. 4.2. Implications on the function of GRP1 and GRP2 The structure and function of GRP1 and GRP2 are still unknown. In these GRPs, tyrosine residues in conserved positions inside the repetitive motif have suggested an involvement of the GRPs in a polymerization process by oxidative cross-linking, i.e., cell wall fortification [3]. In general, GRPs have been attributed to structural cell wall components like HRGPs or PRPs [16,17]. However, this may be an oversimplification, as noted previously [16]. This study demonstrated that GRP1 and GRP2 as well as the ozone-inducible proteins are copper-binding proteins [10]. Thus, it can be reasonably assumed that these GRPs have a common functional role. All these proteins may function as active oxygen scavengers.
4. Discussion 4.1. The Cu2+ complex with 1 (N1Y2G3H4G5G6G7N8Y9G10N11) Peptide 1 likely formed a 1:1 copper complex and exhibited pH-sensitive binding. Potentiomeric measurement for AcGHGG and AcGGHG indicated that two amide nitrogens mainly underwent deprotonation at pH 7.4 [15]. The NMR spectra indicated that the resonances of the His4 Ce1H, His4 Cy2H, His4 ChH, His4 CaH, Gly5 CaH, Asn8 ChH, Asn8 CaH, and Asn11 ChH were significantly broadened (Figs. 6 and 7). From the observations of proton broadening in a pH-dependent fashion, a plausible model for 1 was deduced, as illustrated in Fig. 8, although alternative forms may exist. The 1:1 copper complex could be four-coordinate. Two out of the four copper-binding sites, His4 and Gly5, correspond to His3 and Gly4 in the 2:1 copper complex of the peptide (Y1G2H3G4G5G6Y7) in the ozone inducible proteins [10]. The observation of a similar pattern in the visible CD spectra supports this. The comparison of the visible CD spectra among the three peptides (1, 2 and 3) indicates that it requires the YG segment preceding His4 in 1 for the binding of one copper
Acknowledgements This work was supported in part by a Grant-in-Aid for Sapporo Medical University Foundation for Promotion of Medical Science (to N.M.).
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M. Kamiya et al. / Biochimica et Biophysica Acta 1722 (2005) 69–76 Millhauser, Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy, Biochemistry 39 (2000) 13760 – 13771. C.S. Burns, E. Aronoff-Spencer, C.M. Dunham, P. Lario, N.I. Avdievich, W.E. Antholine, M.M. Olmstead, A. Vrielink, G.J. Gerfen, J. Peisach, W.G. Scott, G.L. Millhauser, Biochemistry 41 (2000) 3991 – 4001. R.P. Bonomo, G. Imperllizzeri, G. Pappalardo, E. Rizzarelli, G. Tabbi, Copper(II) binding modes in the prion octapeptide PHGGGWGQ: a spectroscopic and voltammetric study, Chemistry 6 (2000) 4195 – 4202. A.P. Garnett, J.H. Viles, Copper binding to the octarepeats of the prion protein. Affinity, specificity, folding, and cooperativity: insights from circular dichroism, J. Biol. Chem. 278 (2003) 6795 – 6802. M. Kamiya, Y. Kumaki, K. Nitta, T. Ueno, Y. Watanabe, K. Yamada, T. Matsumoto, K. Hikichi, N. Matsushima, Copper binding to plant ozone-inducible proteins (OI2-2 and OI14-3), Biochem. Biophys. Res. Commun. 314 (2004) 908 – 915. L. Braunschweiler, R.R. Ernst, Coherence transfer by isotropic mixing-application to proton correlation spectroscopy, J. Magn. Reson. 53 (1983) 521 – 528.
[12] M. Piotto, V. Saudek, V. Sklena´r, Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions, J. Biomol. NMR 2 (1992) 661 – 665. [13] L.A. Day, Circular dichroism and ultraviolet absorption of a deoxyribonucleic acid binding protein of filamentous bacteriophage, Biochemistry 12 (1973) 5329 – 5339. [14] Y. Kumaki, K. Nitta, K. Hikichi, T. Matsumoto, N. Matsushima, Side chain-side chain interactions of arginine with tyrosine and aspartic acid in Arg/Gly/Tyr-rich domains within plant glycine-rich RNA binding proteins, J. Biochem. (Tokyo) 136 (2004) 29 – 37. [15] M. Orfei, M.C. Alcaro, G. Marcon, M. Chelli, M. Ginanneschi, H. Kozlowski, J. Brasun, L. Messori, Modeling of copper(II) sites in proteins based on histidyl and glycyl residues, J. Inorg. Biochem. 97 (2003) 299 – 307. [16] G. Sachetto-Martins, L.O. Franco, D.E. de Oliveira, Plant glycine-rich proteins: a family or just proteins with a common motif ? Biochim. Biophys. Acta 1492 (2000) 1 – 14. [17] B. Keller, Structural cell wall proteins, Plant Physiol. 101 (1993) 1127 – 1130.
Regular paper
The binding of copper ions to glycine-rich proteins (GRPs) from Cicer arietinum Masakatsu Kamiyaa,b, Yasuhiro Kumakia, Katsutoshi Nittaa, Takeshi Matsumotob, Kunio Hikichib, Norio Matsushimab,* a
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan b School of Health Sciences, Sapporo Medical University, Sapporo 060-8556, Japan Received 21 August 2004; received in revised form 10 November 2004; accepted 11 November 2004 Available online 10 December 2004
Abstract Cicer arietinum GRP1 and GRP2 are rich in glycine interposed with histidine and tyrosine. In order to study whether or not these proteins bind Cu2+, circular dichroism (CD) and nuclear magnetic resonance (NMR) were measured for three synthetic peptides corresponding to sections of the protein’s sequences including 1, N1Y2G3H4G5G6G7N8Y9G10N11, where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH2 group at the C-terminus. The visible CD spectra for 1 showed a positive peak near 590 nm not at pH 6.0 but pH 7.4 in the presence of copper ions. The Cu2+ binding induced a drastic change in the far-UV CD spectra, showing the occurrence of large conformation changes. In the 2D TOCSY NMR spectra at pH 7.4, the addition of small amounts of CuSO4 caused a significant broadening of proton resonances of not only His4 but also Gly5, Asn8 and Asn11. CD titration experiment suggested that NYGHGGGNYGN including one repeat unit comprises the fundamental Cu2+ binding unit. D 2004 Elsevier B.V. All rights reserved. Keywords: Glycine-rich protein; Copper binding; Histidine/glycine/tyrosine-rich domain; CD; NMR
1. Introduction GRP1 and GRP2 proteins from Cicer arietinum belong to the family of glycine-rich proteins (GRPs) [1]. mRNAs encoding GRP1 and GRP2 accumulate in chickpea plants in response to fungal infection and other stress factors such as wounding and drought treatment [1]. However, the structure and function of GRP1 and GRP2 are still unknown. GRPs have been implicated in structural cell wall components like hydroxyproline-rich glycoproteins (HRGPs) and prolinerich proteins (PRPs) [1–3]. GRP1, with 148 amino acids (PIR accession no. T09527), and the GRP2 fragment, presumably lacking a part of the N-terminal domain (EMBL accession no. AJ01289), show the same basic pattern: an N-terminal region with structural similarities to putative cell wall signal * Corresponding author. Tel.: +81 11 611 2111; fax: +81 11 612 3617. E-mail address: [email protected] (N. Matsushima). 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.11.012
peptides of GRPs from other plants followed by a region of glycine-rich repeats (Fig. 1). The glycine-rich region is rich in glycine interposed with histidine and tyrosine (HGY-rich domain). The HGY-rich domain contains tandem repeats of heptapeptide GGGNYG(H/N) (Fig. 1). The heptapeptide repeats 10 in GRP1, whereas GRP2 has 17 repeats. The HGY/W-rich domains are present in the ozoneinducible proteins OI2-2 and OI14-3 from Atriplex canescens, and mammalian prions that are responsible for the transmissible spongiform encephalopathies [4], as seen in C. arietinum GRPs. The HGY/W-rich domains in ozone-inducible proteins and prions contain tandem repeats of hexapeptide YGHGGG and octapeptide PHGGGWGQ, respectively. These HGY/W-rich domains have shown to bind copper ions [5–10]. However, the Cu2+-binding unit differs from each other. One copper ion is bound to each octapeptide repeat in prions [6,7], while likely two hexapeptide repeats occur in ozone-inducible proteins [10].
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80Ts column (Tosoh Coop. Japan). Finally, all peptides were chemically blocked with an acetyl group (Ac) at the Nterminus and an -NH2 group at the C-terminus. Blocking the N-terminus is important since polypeptide N-terminal amines can bind directly to copper ions. 2.2. Circular dichroism (CD)
Fig. 1. Schematic diagram of the amino acid sequence of glycine-rich proteins GRP1 and GRP2 from Cicer arietinum (A) and the tandem repeats of hexapeptide GGGNYG(H/N) (B). The signal peptide is hatched, and the glycine-rich domain, which is largely different from the consensus sequence, is dotted.
All CD spectra were recorded on a JASCO J-725 spectropolarimeter (Jasco, Japan). The peptide solutions were concentrated to 0.1 mM (for Far-UV CD) and 0.4 mM (for visible CD) in 10 mM phosphate buffer at 25 8C. Two pHs were prepared: pH 6.0 and 7.4. Individual peptide solutions were prepared in the absence of Cu2+ and presence of Cu2+ with a molar ratio of 1:1. Typically, a cell with a 1.0-mm pathlength was used to obtain spectra between 190 and 260 nm with sampling points every 0.2 nm. The average of the five points was plotted. A 1-cm pathlength cell was used for data between 300 nm and 800 nm, also with 0.5-nm sampling intervals. The average of the four points was plotted. Direct CD measurements (h, in mdeg) were converted to molar ellipticity [h] using the relationship [h]=h/cln in 190–260 nm and h/cl in 300–800 nm, where c is the peptide concentration, l is the path length and n is the number of peptide bonds in the sequence. Molar ellipticity [h] is expressed in units deg cm2 dmol 1. 2.3. NMR spectroscopy
This leads to the hypothesis that the repetitive HGY-rich domains in C. arietinum GRPs may also bind copper ions. If so, what the segment of the Cu2+-binding unit is? In order to verify this, circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements were performed using synthetic peptides corresponding to sections of the sequences of GRPs. The present results demonstrate that N1Y2G3H4G5G6G7N8Y9G10N11 binds one Cu2+.
All NMR spectra were measured on JEOL ALPHA 500 or 600 spectrometers. Sequence-specific assignments of
2. Materials and methods 2.1. Peptide samples Considering the experimental observations on copper binding to the synthetic peptides (including YGHGGGY and HGGGY) corresponding to sections of the sequences of ozone-inducible proteins [10], three peptides for experimental studies on Cu2+-binding were designed. These three synthesized peptides correspond to sections of the protein’s sequences that are truncated and/or frame-shifted analogues of the basic repeating units: 1 (NYGHGGGNYGN), 2 (HGGGNY) and 3 (HGGGNYGN). The three peptides were purchased from SIGMA Genosys Japan K.K. (Ishikari, Japan). All peptides were chemically synthesized using solid-phase Fmoc chemistry on an Applied Biosystems Japan Ltd Pioneer peptide synthesizer. Synthetic peptides were purified by reverse-phase HPLC using a TSKgel ODS-
Fig. 2. Far-UV CD (190–260 nm) of peptide 1 (Ac-NYGHGGGNYGNNH2). Peptide concentrations are 0.1 mM in 10 mM phosphate buffer at pH 7.4, 25 8C. The figure shows the CD spectra with the addition of Cu2+ in 0.0 (o), 0.2 (5), 0.4 (w), 0.6 (), 0.8 (+) and 1.0 ( ) mole-equivalents of CuSO4.
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proton resonances for only peptide 1 were obtained by 2D TOCSY (mixing time=80 ms) and 2D ROESY (mixing time=250 ms) experiments [11,12]. The 2D NMR spectra were measured in the phase-sensitive mode using TPPIState for quadrature detection in f1. The water signal was suppressed by the DANTE method in TOCSY and ROESY [12]. Peptide concentrations were 3.5 mM in H2O/D2O (90%/10%), 10 mM phosphate buffer and 0.5 mM NaN3, pH 6.0 at 10 8C and 2.1 mM in pH 7.4. The spectra of 2D TOCSY (mixing time=80 ms) at pH 7.4 were also measured with the addition of Cu2+ in 0.008, 0.034 and 0.064 moleequivalents of CuSO4. The 1D 1H-NMR spectra was performed in 2.5 mM in H2O/D2O (90%/10%), pH. 7.4, 10 mM phosphate buffer and 0.5 mM NaN3 at 10 8C. CuSO4 was added in aliquots of 0.002 mole-equivalents up to 0.034 mole-equivalents. DSS was added to the solutions as an internal reference (0.0 ppm).
3. Results 3.1. Far-UV CD spectra The far-UV CD spectra for peptide 1 were compared at pH 6.0 and pH 7.4 in the absence and the presence of Cu2+ ions. In the absence of Cu2+, the spectrum was unaffected by pH (data not shown). However, the addition of Cu2+ revealed a remarkable, pH-dependent structural change (Fig. 2). A large conformational change was observed in the presence of Cu2+ at pH 7.4; the CD spectrum was dominated by two, new positive peaks near 207 nm and 226 nm in which the latter has been attributed to aromatic groups of tyrosyl residues [6,13,14] (Fig. 2). These two peaks are comparable to positive peaks at 200 nm and 229 nm observed for YGHGGGY in the ozone-inducible proteins, respectively [10]. The large, drastic change in the
Fig. 3. Visible CD spectrum (300–800 nm) of the three peptides, 1 (AcNYGHGGGNYGN-NH2) (A), 2 (Ac-HGGGNY-NH2) (B) and 3 (AcHGGGNYGN-NH2) (C). Peptide concentrations are 0.4 mM in 10 mM phosphate buffer at 25 8C. o, the CD spectra with the addition of Cu2+ in 1.0 mole-equivalents of CuSO4 at pH 7.4; , the CD spectra under the same conditions at pH 6.0.
.
Fig. 4. Direct Cu2+ binding curves for peptide 1 (Ac-NYGHGGGNYGNNH2). Change in [h] with Cu2+ was at 592 nm.
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CD spectra by the addition of Cu2+ indicates that peptide 1 binds Cu2+ at pH 7.4. 3.2. Visible CD spectra When Cu2+ is added to the peptides, CD bands arising due to d–d transitions appear in the visible region. These transitions become observable by CD only when Cu2+ is in a chiral ligand environment, and thus trace amounts of aqueous Cu2+ ions are not detected. The CD spectra for the three peptides, 1, 2 and 3, at pH 6.0 and pH 7.4 are shown in Fig. 3. The binding of Cu2+ to the three
peptides exhibited pH-sensitivity. At pH 6.0, CD bands arising due to d–d transitions were not observed regardless of the presence of Cu2+ (Fig. 3). In contrast, the characteristic CD bands were observed in the presence of Cu2 at pH 7.4. For peptide 1, the CD spectrum had a positive peak near 590 nm with a molecular ellipticity equal to +3.15103, while in the CD spectra peptide 2 and 3 appeared to show very weak peak near 590 nm. The ellipticity for 1 (NYGHGGGNYGN) was very comparable to that of YGHGGGY, YGHGGGYGHGGGY, PHGGGWGQ and HGGGW [6,8,10]. Two other peaks were also observed: a
Fig. 5. Fingerprint region and the ChH region of the 2D-TOCSY spectra and fingerprint regions of the 2D-ROESY spectra of peptide 1 (Ac-N1-Y2-G3-H4-G5G6-G7-N8-Y9-G10-N11-NH2). The 2D-TOCSY spectra is in the upper figure and the 2D-ROESY spectra is in the lower figure. The spectra at pH 6.0 and pH 7.4 are shown in black and red, respectively.
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positive peak near 320 nm due to charge transfer and a negative peak near 480 nm (Fig. 3), while PHGGGWGQ and HGGGW showed a positive peak at 340 nm and a negative band near 720 nm [6,9]. The visible CD spectra observed here clearly indicate that peptide 1 at least binds Cu2+. 3.3. CD titration The CD signals were obtained as a function of titrated Cu2+ to determine the stoichiometry of metal ion to the peptide binding (Fig. 4). The positive CD signal intensity was followed as a function of added Cu2+ at pH 7.4. The titration curve for peptide 1 is shown in Fig. 4. The curve appears to show curvature in the region of about 1.0 added equivalent. The binding curves may be influenced by conformational change dependent on the copper concentration.
resonances linked to the paramaganetic centers by through-bond (contact) or through-space (pseudocontact) interactions. The low levels of Cu2+ in the 1H NMR study enable the observation of significant differential broadening of the resonances of protons close to the copper binding site, as long as the exchange rate of the copper is rapid. The effects of Cu2+ on the 1H NMR spectrum of peptide 1 were studied to determine the nature of the copper-binding sites. The first step of this study was the identification of individual amino acid residues and the sequential assignment in the absence of Cu2+ ions. The identification and assignments were completed unambiguously by the TOCSY and ROESY experiments at pH 6.0 (Fig. 5A,B). Next, the proton assignment at pH 7.4 was performed using the following two procedures: (1)
3.4. NMR assignments (2) The slow electron spin relaxation of the paramagnetic Cu2+ results in marked broadening of proton NMR
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The identification and assignments at pH 7.4 were completed by the comparison the TOCSY spectra at pH 6.0 and pH 7.4 (Fig. 5A). The 1D spectrum at pH 7.4 was compared with the 2D spectrum at pH 7.4 and the 1D and 2D spectra at pHs 6.0 and 7.4. The spectral assignment of the five
Fig. 6. The 2D-TOCSY spectra showing the effects of the addition of Cu2+ to peptide 1 (Ac-N1-Y2-G3-H4-G5-G6-G7-N8-Y9-G10-N11-NH2) (2.1 mM in 90%/10% H20/D2O, pH 7.4, and 10 mM phosphate buffer at 10 8C. The spectra in the absence of Cu2+ are shown in black while the spectra with the addition of Cu2+ in 0.064 mole-equivalent of CuSO4 are shown in red. In A–C, squared cross peaks are observed in the absence of Cu2+.
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glycine residues was performed using a fitting program (http://nakamura-2.ees.hokudai.ac.jp/NakamuraX/nmr/ nmr.html).
In the 1D spectra at pH 7.4, the side chain protons (Cy2H, C H, and CaH) of His4 were unambiguously assigned (Fig. 7A,B). The backbone CaH protons of the five glycine E1
Fig. 7. 1H NMR plots showing the effects of the addition of Cu2+ to peptide 1 (Ac-N1-Y2-G3-H4-G5-G6-G7-N8-Y9-G10-N11-NH2) (2.5 mM in 90%/10% H2O/D2O, pH 7.4, and 10 mM phosphate buffer at 10 8C. (A) 6.5–8.0 ppm; (B) 2.5–3.3 ppm; (C) 3.7–4.1 ppm. The upper, middle and bottom of the figure shows the spectra in the absence of Cu2+ and with the addition of Cu2+ in 0.008 and 0.034 mole-equivalents of CuSO4, respectively.
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residues (Gly3, Gly5, Gly6, Gly7 and Gly10) were also reasonably assigned (Fig. 7C). 3.5. Cu2+ complexation studies of peptide 1 by 1H NMR The addition of small amounts of CuSO4 to 1 caused differential broadening of resonances, which indicated the location of copper binding. The effects of Cu2+ addition on the 2D-TOCSY and 1D spectra of 1 are shown in Figs. 6 and 7. There was a general increase in the linewidth of most resonances with increasing Cu2+ concentration. After the addition of 0.064 mole-equivalents of Cu2+, the resonances of the backbone CaH protons of His4, Gly5 and Asn8, and the ChH protons of Asn8 and Asn11 were so broad that they were indistinguishable from the baselines, whereas other resonances remained relatively sharp (Fig. 6A–C). The significant broadening of the His4 CaH resonance was observed even after the addition of 0.034 mole-equivalent. After this addition, the side chain protons (ChH, CE1H and Cy2H) of His4 were also broadened significantly more than other resonances (Figs. 7A,B). The Gly5 CaH resonance was broadened more strongly than those of the other glycine residues (Fig. 7C), which is consistent with the differential broadening observed in the 2D-TOCSY spectra (Fig. 6B). Moreover, the CE1H and Cy2H protons of His4 in the presence of Cu2+ were downfield from those in the absence of Cu2+. Such downfield shifts are consistent with the observed change in the far-UV CD spectra, indicating structural change induced by the binding of Cu2+ to 1.
Fig. 8. Plausible structure for the complex of Cu2+ with peptide 1 (Ac-N1Y2-G3-H4-G5-G6-G7-N8-Y9-G10-N11-NH2).
ion (Fig. 3). Thus, it is suggested that the NYGHGGGNYGN segment comprises the Cu2+-binding unit. This binding unit predicts that GRP1 and GRP2 bind at least four and eight copper ions per protein, respectively. 4.2. Implications on the function of GRP1 and GRP2 The structure and function of GRP1 and GRP2 are still unknown. In these GRPs, tyrosine residues in conserved positions inside the repetitive motif have suggested an involvement of the GRPs in a polymerization process by oxidative cross-linking, i.e., cell wall fortification [3]. In general, GRPs have been attributed to structural cell wall components like HRGPs or PRPs [16,17]. However, this may be an oversimplification, as noted previously [16]. This study demonstrated that GRP1 and GRP2 as well as the ozone-inducible proteins are copper-binding proteins [10]. Thus, it can be reasonably assumed that these GRPs have a common functional role. All these proteins may function as active oxygen scavengers.
4. Discussion 4.1. The Cu2+ complex with 1 (N1Y2G3H4G5G6G7N8Y9G10N11) Peptide 1 likely formed a 1:1 copper complex and exhibited pH-sensitive binding. Potentiomeric measurement for AcGHGG and AcGGHG indicated that two amide nitrogens mainly underwent deprotonation at pH 7.4 [15]. The NMR spectra indicated that the resonances of the His4 Ce1H, His4 Cy2H, His4 ChH, His4 CaH, Gly5 CaH, Asn8 ChH, Asn8 CaH, and Asn11 ChH were significantly broadened (Figs. 6 and 7). From the observations of proton broadening in a pH-dependent fashion, a plausible model for 1 was deduced, as illustrated in Fig. 8, although alternative forms may exist. The 1:1 copper complex could be four-coordinate. Two out of the four copper-binding sites, His4 and Gly5, correspond to His3 and Gly4 in the 2:1 copper complex of the peptide (Y1G2H3G4G5G6Y7) in the ozone inducible proteins [10]. The observation of a similar pattern in the visible CD spectra supports this. The comparison of the visible CD spectra among the three peptides (1, 2 and 3) indicates that it requires the YG segment preceding His4 in 1 for the binding of one copper
Acknowledgements This work was supported in part by a Grant-in-Aid for Sapporo Medical University Foundation for Promotion of Medical Science (to N.M.).
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