JXB Advance Access originally published online on August 30, 2005
Journal of Experimental Botany 2005 56(420):2695-2703; doi:10.1093/jxb/eri262
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Metal binding by citrus dehydrin with histidine-rich domains
Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
* To whom correspondence should be addressed. Fax: +81 54 238 4881. E-mail: masahara{at}agr.shizuoka.ac.jp
Received 2 December 2004; Accepted 9 July 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Dehydrins are hydrophilic proteins that are responsive to osmotic stress, such as drought, cold, and salinity in plants. Although they have been hypothesized to stabilize macromolecules in stressed cells, their functions are not fully understood. Citrus dehydrin, which accumulates mainly in response to cold stress, enhances cold tolerance in transgenic tobacco by reducing lipid peroxidation. It has been demonstrated that citrus dehydrin scavenges hydroxyl radicals. In this study, the metal binding of citrus dehydrin is reported and the specific domain responsible is identified. The metal binding property of citrus dehydrin was tested using immobilized metal ion affinity chromatography (IMAC). Fe3+, Co2+, Ni2+, Cu2+, and Zn2+ bound to citrus dehydrin, but Mg2+, Ca2+, and Mn2+ did not. Among the bound metals, the highest affinity was detected for Cu2+-dehydrin binding, which showed a dissociation constant of 1.6 µM. Citrus dehydrin was able to bind up to 16 Cu2+ ions. IMAC indicated that His residues contributed to Cu2+-dehydrin binding. The amino acid sequence of CuCOR15 was divided into five domains, of which domain 1 bound Cu2+ most strongly. One portion of domain 1, HKGEHHSGDHH, was the core sequence for the binding. These results suggest that citrus dehydrin binds metals using a specific sequence containing His. Since citrus dehydrin is a radical-scavenging protein, it may reduce metal toxicity in plant cells under water-stressed conditions.
Key words: Citrus unshiu Marcov., cold stress, dehydrin, LEA proteins, metal binding, osmotic stress
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Numerous studies on osmotic stress from drought, cold, and high salinity have been performed, since these abiotic forms of stress are major limiting factors of plant growth (Bray et al., 2000
). Osmotic stress up-regulates many genes, including those for radical-scavenging enzymes, water channels, enzymes for the biosynthesis of osmoprotectants, proteinases, transcription factors, protein kinases, and late embryogenesis abundant (LEA) proteins (Seki et al., 2002; Bray, 2004). Of these, genes characterized well in terms of function have been extensively examined as to the relationship between gene expression and environmental stimuli. However, the functions of many genes, including those for LEA proteins, are not fully understood. There is a need to investigate the functions of these genes to elucidate the osmotic stress response in plants.
Dehydrins are group 2 LEA proteins. They are distributed not only in higher plants but also in algae, yeast, and cyanobacteria (Close, 1996; Ingram and Bartels, 1996; Svensson et al., 2002). Drought, cold, salinity, and the administration of abscisic acid (ABA), all promote the gene expression and accumulation of dehydrin protein (Close, 1997; Svensson et al., 2002). Wounding and radical treatments also induce dehydrin genes (Desikan et al., 2000; Richard et al., 2000). Dehydrins are expressed during the late stages of embryogenesis (Close, 1996). Many copies of dehydrins or dehydrin-related proteins are located in plant genomes. Five kinds of dehydrins in Arabidopsis show distinct tissue-specific distributions and different responses to cold, salt, and ABA (Nylander et al., 2001). Dehydrins have been found to accumulate in the cytoplasm, nucleus, plasma membrane, vacuole membranes, and mitochondria (Close, 1996; Danyluk et al., 1998; Heyen et al., 2002; Hara et al., 2003). Dehydrins are hydrophilic because of their high proportion of Gly and hydrophilic residues. Single or multiple Lys-rich motifs (consensus sequence: EKKGIMDKIKEKLPG) are conserved in all dehydrins (Close, 1996). The Lys-rich motif is believed to form an amphiphilic 
-helix that interacts with cellular macromolecules (Close, 1996, 1997; Soulages et al., 2003). Phospholipids, detergents, and low temperatures enhanced the conformational transition of the Lys-rich motif to form amphiphilic alpha-helices (Ismail et al., 1999b; Soulages et al., 2003). Koag et al. (2003) demonstrated the binding of maize dehydrin to lipid vesicles.
Because dehydrin is strongly induced by cold stress in many plants, the relationship between the accumulation of dehydrin and the enhancement of cold tolerance has been studied. Dehydrin loci and tolerance to chilling were markedly cosegregated in cowpeas (Ismail et al., 1999a). Transgenic plants expressing dehydrins showed higher cold tolerance than the wild types (Kaye et al., 1998; Hara et al., 2003; Puhakainen et al., 2004). It is suggested that the expression of dehydrin genes plays an important role in increasing cold tolerance in plants. Citrus (Hara et al., 2001), peach (Wisniewski et al., 1999), wheat (Houde et al., 1995), and spinach (Kazuoka and Oeda, 1994) dehydrins show cryoprotective effects on freezing-sensitive enzymes such as lactate dehydrogenase. Antifreeze activity has been demonstrated in peach dehydrin (Wisniewski et al., 1999). The data from in vitro experiments indicate that the dehydrin protein can function as a protectant that reduces the degree of cellular injury at low temperatures. However, studies on dehydrin function during stress, other than cold stress, have been rare.
It has recently been reported that transgenic tobacco expresses citrus dehydrin with fewer peroxidized lipids than the wild type when incubated at low temperatures (Hara et al., 2003). Citrus dehydrin shows scavenging activity against hydroxyl radicals and peroxyl radicals (Hara et al., 2004). During the process of scavenging by citrus dehydrin, Gly, Lys, and His residues are markedly degraded, suggesting that the three are involved in the scavenging of radicals (Hara et al., 2004). This antioxidative activity may be a crucial function of dehydrin, because dehydrins are produced not only by cold but also by drought and salinity, both of which are environmental stimuli which generate radicals in plants (McKersie et al., 1993; Shen et al., 1997; Iturbe-Ormaetxe et al., 1998).
It is postulated that hydroxyl radicals, which are extremely cytotoxic, are generated during the response to water stress in plants (Iturbe-Ormaetxe et al., 1998). The hydroxyl radical is generated by the metal-catalysed Haber–Weiss reaction in which transition metals, such as Fe and Cu, participate (Halliwell and Grootveld, 1988). In both transition states, i.e. as Fe2+ and Fe3+ or Cu+ and Cu2+, these metals can form hydroxyl radicals by reacting with hydrogen peroxide or superoxide anions. It is known that some antioxidative proteins, such as metallothionein, ceruloplasmin, and serum albumin, bind metal ions to stabilize them (Soriani et al., 1994; Vasak and Hasler, 2000; Kang et al., 2001; Akashi et al., 2004). Indeed, it is reported that some dehydrins can bind metal ions (Svensson et al., 2000; Kruger et al., 2002; Alsheikh et al., 2003; Herzer et al., 2003). Svensson et al. (2000) noted that His residues in dehydrins could be involved in dehydrin-metal binding, because some dehydrins are rich in His, which is one form of metal-affinity residue. Ricinus communis and Glycine max dehydrins could be retained on a Cu2+-charged chelating column; they were eluted with imidazole, which is an analogue of His (Kruger et al., 2002; Herzer et al., 2003). These results suggest a role for His in dehydrin metal binding. However, to confirm the role of His in the metal binding of dehydrins, more circumstantial evidence is needed on the contribution of His and specific metal-binding domains. In this paper, it has been analysed whether citrus dehydrin can bind metals, and if so, which domains are involved.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cloning of CuCOR15
cDNAs for CuCOR15 (Citrus unshiu cold-regulated 15 kDa) protein were obtained by screening a cDNA library derived from the flavedo tissue of C. unshiu (Hara et al., 1999
). The library, consisting of 4.5x105 recombinants, was screened using the plaque hybridization method. The probe used for the screening was full-length CuCOR19 cDNA. The CuCOR19 (C. unshiu cold-regulated 19 kDa) protein is a dehydrin of C. unshiu which has three Lys-rich motifs (Hara et al., 1999). Hybridization and detection were performed using a DIG DNA Labeling and Detection Kit (Roche Diagnostics, Tokyo). More than 100 positive clones were obtained and 20 randomly-selected clones were sequenced. Ten clones were full-length clones and encoded the same CuCOR15.
Recombinant expression of CuCOR15
Recombinant proteins were obtained by using the pET22b Escherichia coli expression system (Novagen, WI). A pelB leader was attached at the N terminus of the recombinant proteins to transport them to the periplasm of the host bacteria. The His–Tag sequence was not used in this experiment. For the expression of the entire open reading frame (ORF) of CuCOR15 protein, the corresponding cDNA was obtained by a polymerase chain reaction (PCR) with a sense primer containing a BamHI site (5'-AATTCGGATCCGATGTCAGGAGTTATTCACAAG-3', corresponding amino acid sequence: MSGVIHK) and an antisense primer containing a HindIII site (5'-GCGGCCGCAAGCTTTTAATCGCTGTCACTGCT-3', corresponding amino acid sequence: SSDSD and a stop codon). The cDNA fragment was inserted into the BamHI and HindIII sites of the pET22b vector to form an in-frame junction between the pelB leader and the ORF of CuCOR15. A construct expressing the region from Leu12 to the C terminus in the CuCOR15 ORF was prepared in a similar way. The cDNA was amplified by PCR with a sense primer containing a BamHI site (5'-AATTCGGATCCCCCCCTTCACATGGGAGGAGGGCAA-3', corresponding amino acid sequence: LHMGGGQ) and the same antisense primer described above. The PCR product was inserted into the BamHI and HindIII sites of pET22b. An expression vector for a mutagenized CuCOR15 lacking domains 4 and 5 was produced by introducing a stop codon into the expression vector containing the partial CuCOR15 ORF from Leu12 to the C terminus. Site-directed mutagenesis was applied to form a TAA stop codon flanking the N-terminus side of domain 4 (Gly105) by using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, CA). The mutation introduced was confirmed by DNA sequencing.
Escherichia coli strain BL21 containing each expression construct was precultured at 37 °C. One-millimolar isopropyl ß-D-thiogalactopyranoside was added to the culture. After incubating for 3 h at 28 °C, the periplasmic fraction was obtained according to the manufacturer's instructions (Novagen, WI). Bacterial cells obtained from the culture (950 ml) were resuspended in 4 ml of the culture medium. Chloroform (1.2 ml) and 10 mM TRIS–HCl buffer pH 8 (6 ml) were added to the cell suspension. After mixing, the suspension was centrifuged at 12 000 g for 10 min at 4 °C. A supernatant was used as a periplasmic fraction. The fraction was heated at 85 °C for 10 min. After centrifugation at 12 000 g for 10 min at 4 °C, the supernatant was adjusted to 70% saturation with ammonium sulphate and then centrifuged at 12 000 g for 20 min at 4 °C. The pellet was dissolved and desalted on a Sephadex G-25 column (Amersham Pharmacia Biotech, Tokyo) equilibrated with 5 mM potassium phosphate buffer (pH 7.0). The sample was next subjected to DEAE-Toyopearl 650M (Tosoh, Tokyo) column chromatography. The protein was eluted with an NaCl gradient (0 M to 1 M). At this step, the purity of the protein was approximately 98% according to the signal intensity of bands in the SDS–PAGE gel stained with Coomassie Brilliant Blue. The sample was desalted using the Sephadex G-25 column and freeze-dried. The dried sample was weighed and kept at –20 °C prior to use.
MS analysis
The molecular mass of the recombinant CuCOR15 protein from Leu12 to the C terminus was determined by electrospray ionization/mass spectrometry (ESI/MS, API-150EX, Applied Biosystems/MDS SCIEX, Toronto). The purified protein was dissolved in 5 mM potassium phosphate buffer at pH 7.0. Twenty-five microlitres of the protein solution (4 mg ml–1, 267 µM) and 75 µl of 10 mM ammonium acetate at pH 6.8 were combined to prepare an injection sample. The positive ion ESI mass spectra were recorded by averaging 80 scans from m/z 400–3000 with a pause of 5 ms. The minimum molecular mass was determined from the deconvoluted spectra.
Diethyl pyrocarbonate (DEPC) treatment
A recombinant CuCOR15 protein from Leu12 to the C terminus was dissolved in 100 mM sodium phosphate buffer at pH 6.0 at a concentration of 1.3 mg ml–1 (86.7 µM). The protein solution (160 µl) was combined with an appropriate concentration of DEPC solution (40 µl) dissolved in 10% ethanol to give a final concentration of DEPC of 4 mM or 20 mM. The ratio of modification was determined by the absorbance of the ethoxyformyl group at 240 nm. The molar extinction coefficients of the ethoxyformyl group at the DEPC concentrations of 4 mM and 20 mM were 4.0x103 and 6.2x103 M–1 cm–1, respectively (Roosemont, 1978).
Metal chelating affinity chromatography
Interactions between metal ions and polypeptides were judged by means of immobilized metal ion affinity chromatography (IMAC) using HiTrap Chelating HP (Amersham Pharmacia Biotech, Tokyo) according to Ueda et al. (2003). The columns were charged by applying 3 ml of 100 mM MgCl2, CaCl2, MnCl2, FeCl3, CoCl2, NiCl2, CuCl2, and ZnCl2, respectively. After washing out the excess metal with 5 ml of deionized water, the column was equilibrated with 50 mM TRIS–HCl buffer at pH 7.4 containing 1 M NaCl (EQ buffer). One-hundred microlitres of a protein solution (a recombinant CuCOR15 protein from Leu12 to the C terminus, 1 mg ml–1, 66.7 µM) was applied to the column. The unbound recombinant protein was washed out with 12 ml of the EQ buffer followed by 5 ml of water. The bound protein was eluted by the addition of 5 ml of 250 mM EDTA. One millilitre of each was fractionated and subjected to SDS–PAGE analysis. A HiTrap Chelating HP column charged with no metal was used as a control.
To determine which amino acids were involved in metal-binding, elution was carried out with each amino acid, i.e. Gly, Ala, Val, Leu, Ile, Met, Pro, Ser, Thr, Asn, Gln, Asp, Glu, Lys, His, Arg, and Tyr. One-hundred microlitres of a protein solution (a recombinant CuCOR15 protein from Leu12 to the C terminus, 1 mg ml–1, 66.7 µM) was loaded into the Cu2+-chelating column. After washing the column with 7 ml of the EQ buffer, 5 ml of each amino acid dissolved in the EQ buffer was added. The amino acid concentrations were 150 mM and 20 mM for amino acids except Tyr and for Tyr, respectively. The column was then washed with 250 mM EDTA. The fraction size was 1 ml. The fractions were analysed by SDS–PAGE.
The degree of metal–polypeptide binding was compared using the method of Chen et al. (1998) with slight modification. A recombinant CuCOR15 protein from Leu12 to the C terminus, DEPC-treated recombinant CuCOR15s, a mutagenized recombinant CuCOR15 without domains 4 and 5, domains 1 to 5, and a His-rich sequence of domain 1 were applied to the Cu2+-chelating column. The column was washed with 10 ml of the EQ buffer, and a linear gradient of imidazole (13 ml, from 0 mM to 250 mM in the EQ buffer) was used. The gradient was formed using the EGP system (Bio-Rad, Tokyo). The eluate was fractionated and the volume of each fraction was measured. The imidazole concentration was monitored at 240 nm. Fractions containing recombinant proteins were analysed by SDS-PAGE. The protein amount was estimated from the signal intensity of bands stained with Coomassie Brilliant Blue. Domains 1 to 5 and the His-rich sequence of domain 1 contain Trp at the N-terminus. These were detected by measuring the absorbance at 280 nm.
Metal binding assay
The metal binding of the recombinant CuCOR15 protein from Leu12 to the C terminus was analysed using an ultrafiltration method described by Nishikawa et al. (1997). Mixtures containing 16.7 µM (250 µg ml–1) CuCOR15, 10 mM TRIS–HCl pH 7.4, 100 mM NaCl, and appropriate concentrations of CoCl2, NiCl2, CuCl2, or ZnCl2, were incubated at 4 °C for 10 min. In the case of FeCl3, 10 mM MES–NaOH at pH 5.5 was used. The binding was saturated within 10 min (data not shown). MES–NaOH (pH 5.5, 6.0, and 6.4) and TRIS–HCl (pH 6.8 and 7.4) were used to vary the pH. Ionic strength was changed by the addition of NaCl (up to 1 M). Any insoluble complex was removed by centrifugation at 12 000 g for 5 min at 4 °C. The supernatant was then subjected to ultrafiltration (Ultrafree-MC, 5000 NMWL, Millipore, MA). The apparatus was centrifuged at 4000 g for 30 min at 4 °C. The metal concentration in the filtered solution was measured to determine the free metal concentration (F) as described below. After that, bound metal concentration (B), B/F value, and how many atoms the recombinant protein bound to each sample were calculated. For Scatchard plots, the number of metal atoms (x-axis) was plotted versus the B/F value (y-axis), and appropriate straight lines were drawn.
Concentrations of Co2+, Ni2+, Cu2+, and Zn2+ were determined using a calcein assay (Argirova and Ortwerth, 2003). For the Co2+, Ni2+, and Cu2+ assay, 50 µl of sample solution was added to 3 ml of 1 µM calcein solution in 5 mM TRIS–HCl buffer at pH 6.8. Quenching of fluorescence was detected at an excitation wavelength of 488 nm and emission wavelength at 517 nm. For the Zn2+ assay, the calcein solution was prepared with 0.1 M NaOH, and the increase in fluorescence was monitored under the same conditions described above. The concentration of each metal was calculated from the calibration curves. Linearity of the curves was found in the following ranges: Co2+ (0–30 µM), Ni2+ (0–30 µM), Cu2+ (0–60 µM), and Zn2+ (0–80 µM). Iron was determined using a modified o-phenanthroline method (Tamura et al., 1974). The sample (70 µl) and hydroxylammonium chloride solution (90 µl) were mixed and kept at room temperature for 10 min. One-molar sodium acetate buffer at pH 4 (120 µl) and 5 mM o-phenanthroline solution (120 µl) were added to the mixture. Absorbance at 510 nm was measured. The linearity was maintained from 0 to 300 µM.
Peptide synthesis
Six peptides were prepared by automated solid phase peptide synthesis on a synthesizer (PSSM8, Shimadzu, Kyoto). The sequences of synthetic peptides were as follows. Domain 1: WGGQKEEDKHKGEHHSGDHH, domain 2a: WTTDVHHQQQYHGGEH, domain 3: WKEGLVDKIKQQIPGVG, domain 4: WGGEGAHGEEKKKKKKEKKK, domain 5: WKKHEDGHESSSSSDSD, and the His-rich region of domain 1: WHKGEHHSGDHH. A Trp residue was introduced at the N-terminus of each peptide to allow detection by UV absorption at 280 nm. Synthesized peptides were purified by C18 reversed-phase column chromatography to 98% homogeneity with a 20% to 40% linear gradient of acetonitrile in TFA solution (0.1%) over 20 min. The peptides were identified using an amino acid analyser (L-8500, Hitachi, Tokyo).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Preparation of recombinant citrus dehydrin
The radical scavenging activity of a CuCOR19 (Citrus unshiu cold-regulated 19 kDa protein) (Hara et al., 2003
, 2004) has previously been reported. CuCOR19 belongs to the dehydrin family, since it has Lys-rich motifs which are typical conserved domains of dehydrin (Hara et al., 1999). When a flavedo cDNA library of C. unshiu was screened with a probe for CuCOR19 cDNA, another kind of dehydrin cDNA was isolated. The new clone was identical in amino acid sequence to CuCOR19 except that it had only two Lys-rich motifs. The gene encoded a 15 kDa protein composed of 137 amino acids. Thus the clone was designated CuCOR15. The accession number for CuCOR15 is AB178479. The amino acid sequence of CuCOR15 is shown in Fig. 1A. It is a member of the citrus dehydrin family, since identity at the amino acid level in CuCOR15 versus C. clementina dehydrin (AAQ92310
[GenBank]
) and in CuCOR15 versus C. paradisi dehydrin (AAK52077
[GenBank]
) was 98.6% and 96.4%, respectively. CuCOR15 is very similar to CuCOR19 in that its expression was enhanced by cold stress in citrus and the recombinant protein showed antioxidative activity (data not shown). However, the domain composition of CuCOR15 is simpler than that of CuCOR19. For this reason, subsequent experiments were conducted with CuCOR15.
|
Recombinant CuCOR15 protein was prepared using the pET22b E. coli expression system. Two kinds of expression vectors were constructed: a leader peptide for periplasmic transportation was attached to the N terminus of a full-length open reading frame (ORF) or part of the ORF from Leu12 (underlined in Fig. 1) to the C terminus. Expression succeeded with the latter construction. After digestion of the leader peptide, a short sequence from the vector (MDIGINSDPP) was attached to the N terminus of the partial ORF. Below, this protein is designated as ‘recombinant CuCOR15 protein’ or simply ‘CuCOR15’. SDS-PAGE analysis revealed the size of the recombinant CuCOR15 protein to be far larger than 15 kDa (Fig. 1B). The molecular weight determined by electrospray ionization/mass spectrometry (ESI/MS) and the calculated molecular weight were 15151±2 and 15078, respectively. It is likely that two K+ molecules bound to the recombinant CuCOR15 protein, since the ESI/MS analysis was performed in a potassium phosphate buffer. A previous report noted that some dehydrins showed a higher molecular weight in SDS-PAGE than their calculated molecular weight (Svensson et al., 2000
).
Metal binding property of CuCOR15
Immobilized metal ion affinity chromatography (IMAC) was performed to identify the metals which were bound by the recombinant CuCOR15 protein. IMAC has been used to test the binding affinity between proteins and metal ions (Chaga, 2001; Ueda et al., 2003). CuCOR15 protein was applied to IMAC columns chelating Mg2+, Ca2+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, or Zn2+ under high ionic strength (1 M NaCl). The chromatography was performed at pH 7.4 for all metals except Fe3+. Because Fe3+ in an alkaline solution forms Fe colloids, the pH was adjusted to 5.5 to run the Fe3+-column. CuCOR15 was retained in the columns immobilizing Fe3+, Co2+, Ni2+, Cu2+, and Zn2+, but not in the columns immobilizing no metal, Mg2+, Ca2+, or Mn2+ (Fig. 2). The CuCOR15 retained in the column was eluted with the EDTA wash, indicating that CuCOR15 binds Fe3+, Co2+, Ni2+, Cu2+, and Zn2+.
|
IMAC analysis, however, is only semi-quantitative when used to estimate the binding between CuCOR15 and metals. Thus the binding affinity between the recombinant CuCOR15 protein and metals was compared using the dissociation constant (KD) obtained from the Scatchard plots (Fig. 3). One binding mode was identified for Fe3+ and Co2+ binding, and two for Ni2+, Cu2+, and Zn2+ binding. The highest affinity was found for Cu2+-CuCOR15 binding (KD=1.6 µM) and Ni2+-CuCOR15 binding (KD=1.8 µM) (Fig. 3C, D). Inversely, the Fe3+-CuCOR15 binding showed the lowest affinity (KD=1.4 mM) (Fig. 3A). The order of binding affinity was Cu2+=Ni2+ >Zn2+ >Co2+ >>Fe3+. CuCOR15 was able to bind up to 18 Fe3+, 2 Co2+, 6 Ni2+, 16 Cu2+, and 12 Zn2+ ions. The following experiments were carried out on Cu2+-CuCOR15 binding, due to its high affinity and binding ability to so many Cu2+ ions. Because the binding of CuCOR15 to Cu2+ was not influenced by pH (from 5.5 to 7.4) or ionic strength (up to 1 M NaCl), the binding is likely to be specific and stable under physiological conditions (data not shown).
|
Amino acids associated with the binding of CuCOR15 to Cu2+
To elucidate the binding mechanism, an investigation was made into which amino acid residues contributed to Cu2+-CuCOR15 binding. First, ligand elution in IMAC was carried out with each amino acid comprising CuCOR15 (Fig. 4). The recombinant CuCOR15 lacks Phe, Trp, and Cys, so 17 kinds of amino acids were subjected to elution. Only His could dissociate the binding of CuCOR15 to Cu2+ (Fig. 4A). Other amino acids (Gly, Ala, Val, Leu, Ile, Met, Pro, Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, and Tyr) did not elute CuCOR15 (Fig. 4B). The CuCOR15 retained in the column could be eluted using a gradient of imidazole, which is an analogue of His (Fig. 5). The imidazole concentration at elution was 112 mM. His residues in CuCOR15 were modified by diethyl pyrocarbonate (DEPC). Although the DEPC concentration was elevated to 20 mM, only 8 His residues were modified. The addition of urea to the DEPC reaction did not enhance the efficiency (data not shown). DEPC treatment reduced the strength of Cu2+-CuCOR15 binding, because CuCOR15 treated with 4 mM and 20 mM DEPC was eluted by 60 mM and 50 mM imidazole, respectively (Fig. 5). These results suggest that His is a crucial residue that contributes to Cu2+-CuCOR15 binding.
|
|
Determination of the metal-binding domain in CuCOR15
Based on the disposition of sequence motifs, CuCOR15 was divided into five domains (Fig. 1A). Domain 1 (D1, GGQKEEDKHKGEHHSGDHH) is located near the N-terminus of CuCOR15. Two mutually similar kinds of domain 2 are found in CuCOR15. One domain 2 (D2a, TTDVHHQQQYHGGEH) is near the C-terminus side of domain 1. Another domain 2 (D2b, TTDVHHQQQQQQYHGGEHREGEH) is located between the two Lys-rich motifs designated as domain 3 (D3, KEGLVDKIKQQ/KIPGVG). Domains 4 and 5, respectively, contain the Lys-cluster and the Ser-cluster.
To estimate the relative strength of binding between Cu2+ and each domain, a recombinant CuCOR15 (D1-D2a-D3-D2b-D3-D4-D5), a mutagenized recombinant CuCOR15 lacking domains 4 and 5 (D1-D2a-D3-D2b-D3), each domain from 1 to 5 (D1, D2a, D3, D4, and D5), and the His-rich region of domain 1 (HKGEHHSGDHH) were subjected to IMAC chelating Cu2+. The chromatography was performed as illustrated in Fig. 5. The recombinant CuCOR15, the mutagenized CuCOR15 lacking domains 4 and 5, domain 1, and HKGEHHSGDHH bound Cu2+ to the same extent: the concentration of imidazole at the elution peak was between 106 and 112 mM (Table 1). The binding of domain 2a (imidazole concentration: 92 mM) was slightly weaker than that of domain 1. Imidazole concentrations for the elution of domains 4 and 5 were 50 mM and 58 mM, respectively. Domain 3 was eluted at the void volume. Among the peptides tested, the number of His residues was positively correlated to the intensity of the Cu2+ binding. Because the binding of HKGEHHSGDHH was equal to that of native CuCOR15, HKGEHHSGDHH was a crucial sequence for the binding of Cu2+ in CuCOR15. Domain 2s also appeared to contribute to the binding. On the other hand, it appears likely that domains 4 and 5 did not participate to any great extent, since the binding of these domains was weak and the mutant CuCOR15, lacking in both domains, showed the same binding activity as native CuCOR15. Domain 3 was not associated with the binding of Cu2+.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Dehydrins are proteins which accumulate in plants exposed to osmotic stress. The function of dehydrins, however, remains to be elucidated. Recently, it has been reported that citrus dehydrin is an antioxidative protein (Hara et al., 2003
, 2004). The citrus dehydrin scavenges hydroxyl radicals generated by the metal/H2O2 system (Hara et al., 2004). In mammals, several radical scavenging proteins chelate metals, indicating that the metal binding of protein controls the generation of radicals (Soriani et al., 1994; Vasak and Hasler, 2000; Kang et al., 2001). It has been reported that some dehydrins bind metals (Svensson et al., 2000; Kruger et al., 2002; Alsheikh et al., 2003; Herzer et al., 2003). Four recombinant Arabidopsis dehydrins bound to a Cu2+- and Ni2+-charged chelating column (Svensson et al., 2000). They predicted that His residues in dehydrins could be related to metal binding. The protein, however, was not eluted with 1 M imidazole. Ricinus communis dehydrin-like protein and Glycine max dehydrin could be captured on the Cu2+-charged chelating column (Kruger et al., 2002; Herzer et al., 2003). These dehydrins were eluted with imidazole, but the binding mechanisms were not discussed. Alsheikh et al. (2003) reported that phosphorylation of a Ser-cluster domain is needed for Ca2+ binding. These results show that it remains unclear how dehydrins bind metals, especially transition metals. It has therefore been demonstrated that a citrus dehydrin (CuCOR15) binds metals, and the metal-binding domains have been investigated.
CuCOR15 bound Fe3+, Co2+, Ni2+, Cu2+, and Zn2+. The order of binding affinity was Cu2+=Ni2+ >Zn2+ >Co2+ >>Fe3+. For Ni2+, Cu2+, and Zn2+, two classes of binding (high and low affinity), were found. The number of metal ions bound at high affinity was estimated at about two (Fig. 3C, D, E). This shows that two metal ions may preferentially associate with CuCOR15. Comparing the KD of CuCOR15 and other reported copper-binding proteins, the value for CuCOR15 (1.6 µM) was lower than that for a prion protein (14 µM) (Viles et al., 1999), but higher than that for a brain S100b protein (0.46 µM) (Nishikawa et al., 1997) or a salivary histatin (38 nM) (Gusman et al., 2001). CuCOR15 bound up to 16 atoms of Cu2+ as a result of both high and low affinity binding. The binding capacity of CuCOR15 was much greater than that of the prion protein (4 atoms), the S100b protein (6.5 atoms), or the histatin (4 atoms). Since the Cu2+-binding of CuCOR15 was not influenced by pH or ionic concentration, the binding may remain strong under physiological conditions.
Cys, His, Lys, Arg, Trp, Tyr, Phe, and the N-terminus may be involved in metal–protein interactions (Ueda et al., 2003). CuCOR15 does not contain any Cys, Trp, or Phe residues. The IMAC experiments demonstrated that only His contributes to the metal binding of CuCOR15. In addition, the His-rich sequence in domain 1 (HKGEHHSGDHH) showed a similar binding activity to CuCOR15. These results suggest that HKGEHHSGDHH may be a core sequence that binds metals in CuCOR15. It is noticed that the sequence contains double His (HH) sequences and His-X3-His motifs (HKGEH and HSGDH). The double His sequence binds metals more strongly than a single His (Gusman et al., 2001). The His-X3-His motif has been characterized as a metal binding site in many metal-binding proteins, such as C2H2-type zinc finger transcription factors (Bouhouche et al., 2000), zinc-dependent metallopeptidases (Jongeneel et al., 1989), histatins (Gusman et al., 2001), and synthetic peptides binding metals (Corazza et al., 1999). If a peptide containing the motif forms a helix, two His residues could be located close together on the surface of the coil. Thus the His-X3-His motif shows strong metal binding (Suzuki et al., 1998). The sequence of HKGEHHSGDHH may bind metals by means of similar mechanisms. As the other model, HHSGDHH may form a loop structure similar to Zn finger motifs (Bouhouche et al., 2000), provided the two HHs connecting together can chelate metals. Binding assays using a series of mutant peptides that include deletion mutants or positional mutants of His residues will elucidate the metal binding mode of CuCOR15.
In general, His residues are rare in proteins, with the proportion being approximately 2% of the amino acid content (Ueda et al., 2003). Dehydrins, however, contain a higher proportion of His. In the Arabidopsis genome, there are 10 dehydrin-related proteins. The range of His content was 3.2% to 13.5%. Moreover, 7 dehydrins out of 10 possess the double His sequence and/or the His-X3-His motif (data not shown). These results suggest that most dehydrins may be metal-binding proteins. On the other hand, the Lys-rich motif (domain 3) of CuCOR15 did not bind Cu2+. It has been reported that Lys-rich motifs are crucial for the interaction between dehydrins and macromolecules (Close, 1996; Ingram and Bartels, 1996; Koag et al., 2003; Soulages et al., 2003). Because the metal-binding domain and the macromolecular binding site are located separately, dehydrins may store metal ions and keep them in isolation at specific sites in the cell.
The antioxidative activity of citrus dehydrin can be discussed from two aspects: the scavenging of radicals which have already been generated, and the binding of metals which are sources for radical generation. Dehydration and cold, which promote dehydrin accumulation, are environmental cues producing reactive oxygen species in plants (McKersie et al., 1993; Shen et al., 1997; Iturbe-Ormaetxe et al., 1998). Catalytic metals released from metal–proteins could produce hydroxyl radicals that cause oxidative damage in plants exposed to water stress (Iturbe-Ormaetxe et al., 1998). Because the dehydrated cytoplasm contracts, the catalytic metals may produce more radicals. Dehydrins may reduce the toxicity of metals by binding them and scavenging the radicals that they generate. It is also known that high levels of dehydrins accumulate in the seed embryo. The role of dehydrins in seeds could be to protect the embryo against toxicity from maternally-derived metals concentrated by dehydration.
Metallothioneins and phytochelatins have been a focus of research interest, not only as metal-binding proteins but also as antioxidants in plants (Cobbett, 2000; Hall, 2002). On the other hand, the present results show that citrus dehydrin, which is a radical-scavenging protein, binds metals not via Cys but via His. It is therefore proposed that plants may have two classes of antioxidative metal-binding proteins: an SH-type of metal-binding protein like metallothionein or phytochelatin, and a non-SH-type of metal-binding protein like dehydrin. Both classes may be needed to reduce the oxidative damage induced in plants by environmental stress.
|
Acknowledgements |
|---|
We would like to thank Dr Yamazaki for ESI/MS. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (16780225).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Akashi K, Nishimura N, Ishida Y, Yokota A. 2004. Potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon. Biochemical and Biophysical Research Communications 323, 72–78.[CrossRef][Web of Science][Medline]
Alsheikh MK, Heyen BJ, Randall SK. 2003. Ion binding properties of the dehydrin ERD14 are dependent upon phosphorylation. Journal of Biological Chemistry 278, 40882–40889.
Argirova MD, Ortwerth BJ. 2003. Activation of protein-bound copper ions during early glycation: study on two proteins. Archives of Biochemistry and Biophysics 420, 176–184.[CrossRef][Web of Science][Medline]
Bouhouche N, Syvanen M, Kado CI. 2000. The origin of prokaryotic C2H2 zinc finger regulators. Trends in Microbiology 8, 77–81.[CrossRef][Web of Science][Medline]
Bray EA. 2004. Genes commonly regulated by water-deficit stress in Arabidopsis thaliana. Journal of Experimental Botany 55, 2331–2341.
Bray EA, Bailey-Serres J, Weretilnyk E. 2000. Responses to abiotic stresses. In: Buchanan B, Gruissem W, Jones RL, ed. Biochemistry and molecular biology of plants. MD: American Society of Plant Physiologists, 1158–1203.
Chaga GS. 2001. Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. Journal of Biochemical and Biophysical Methods 49, 313–334.[CrossRef][Web of Science][Medline]
Chen HM, Muramoto K, Yamauchi F, Fujimoto K, Nokihara K. 1998. Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein. Journal of Agricultural and Food Chemistry 46, 49–53.[CrossRef][Web of Science][Medline]
Close TJ. 1996. Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiologia Plantarum 97, 795–803.[CrossRef]
Close TJ. 1997. Dehydrins: a commonality in the response of plants to dehydration and low temperature. Physiologia Plantarum 100, 291–296.[CrossRef]
Cobbett CS. 2000. Phytochelatins and their roles in heavy metal detoxification. Plant Physiology 123, 825–832.
Corazza A, Vianello F, Zennaro L, Gourova N, Di Paolo ML, Signor L, Marin O, Rigo A, Scarpa M. 1999. Enzyme mimics complexing Cu(II) ion: structure–function relationships. The Journal of Peptide Research 54, 491–504.
Danyluk J, Perron A, Houde M, Limin A, Fowler B, Benhamou N, Sarhan F. 1998. Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. The Plant Cell 10, 623–638.
Desikan R, Neill SJ, Hancock JT. 2000. Hydrogen peroxide-induced gene expression in Arabidopsis thaliana. Free Radical Biology and Medicine 28, 773–778.[CrossRef][Web of Science][Medline]
Gusman H, Lendenmann U, Grogan J, Troxler RF, Oppenheim FG. 2001. Is salivary histatin 5 a metallopeptide? Biochimica et Biophysica Acta 1545, 86–95.[CrossRef][Medline]
Hall JL. 2002. Cellular mechanisms for heavy metal detoxification and tolerance. Journal of Experimental Botany 366, 1–11.
Halliwell B, Grootveld M. 1988. Methods for the measurement of hydroxyl radicals in biochemical systems: deoxyribose degradation and aromatic hydroxylation. Methods of Biochemical Analysis 33, 59–90.[Web of Science][Medline]
Hara M, Fujinaga M, Kuboi T. 2004. Radical scavenging activity and oxidative modification of citrus dehydrin. Plant Physiology and Biochemistry 42, 657–662.[Medline]
Hara M, Terashima S, Fukaya T, Kuboi T. 2003. Enhancement of cold tolerance and inhibition of lipid peroxidation by citrus dehydrin in transgenic tobacco. Planta 217, 290–298.[Web of Science][Medline]
Hara M, Terashima S, Kuboi T. 2001. Characterization and cryoprotective activity of cold-responsive dehydrin from Citrus unshiu. Journal of Plant Physiology 158, 1333–1339.[CrossRef]
Hara M, Wakasugi Y, Ikoma Y, Yano M, Ogawa K, Kuboi T. 1999. cDNA sequence and expression of a cold-responsive gene in Citrus unshiu. Bioscience, Biotechnology, and Biochemistry 63, 433–437.[Medline]
Herzer S, Kinealy K, Asbury R, Beckett P, Eriksson K, Moore P. 2003. Purification of native dehydrin from Glycine max cv., Pisum sativum, and Rosmarinum officinalis by affinity chromatography. Protein Expression and Purification 28, 232–240.[CrossRef][Web of Science][Medline]
Heyen BJ, Alsheikh MK, Smith EA, Torvik CF, Seals DF, Randall SK. 2002. The calcium-binding activity of a vacuole-associated, dehydrin-like protein is regulated by phosphorylation. Plant Physiology 130, 675–687.
Houde M, Daniel C, Lachapelle M, Allard F, Laliberte S, Sarhan F. 1995. Immunolocalization of freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues. The Plant Journal 8, 583–593.[CrossRef][Web of Science][Medline]
Ingram J, Bartels D. 1996. The molecular basis of dehydration tolerance in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 377–403.[CrossRef][Web of Science][Medline]
Ismail AM, Hall AE, Close TJ. 1999a. Allelic variation of a dehydrin gene cosegregates with chilling tolerance during seedling emergence. Proceedings of the National Academy of Sciences, USA 96, 13566–13570.
Ismail AM, Hall AE, Close TJ. 1999b. Purification and partial characterization of a dehydrin involved in chilling tolerance during seedling emergence of cowpea. Plant Physiology 120, 237–244.
Iturbe-Ormaetxe I, Escuredo PR, Arrese-Igor C, Becana M. 1998. Oxidative damage in pea plants exposed to water deficit or paraquat. Plant Physiology 116, 173–181.
Jongeneel CV, Bouvier J, Bairoch A. 1989. A unique signature identifies a family of zinc-dependent metallopeptidases. FEBS Letters 242, 211–214.[CrossRef][Web of Science][Medline]
Kang JH, Kim KS, Choi SY, Kwon HY, Won MH. 2001. Oxidative modification of human ceruloplasmin by peroxyl radicals. Biochimica et Biophysica Acta 1568, 30–36.[Medline]
Kaye C, Neven L, Hofig A, Li QB, Haskell D, Guy C. 1998. Characterization of a gene for spinach CAP160 and expression of two spinach cold-acclimation proteins in tobacco. Plant Physiology 116, 1367–1377.
Kazuoka T, Oeda K. 1994. Purification and characterization of COR85-oligomeric complex from cold-acclimated spinach. Plant and Cell Physiology 35, 601–611.
Koag MC, Fenton RD, Wilkens S, Close TJ. 2003. The binding of maize DHN1 to lipid vesicles. Gain of structure and lipid specificity. Plant Physiology 131, 309–316.
Kruger C, Berkowitz O, Stephan UW, Hell R. 2002. A metal-binding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis L. Journal of Biological Chemistry 277, 25062–25069.
McKersie BD, Chen Y, de Beus M, Bowley SR, Bowler C, Inzé D, D'Halluin K, Botterman J. 1993. Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.). Plant Physiology 103, 1155–1163.[Abstract]
Nishikawa T, Lee IS, Shiraishi N, Ishikawa T, Ohta Y, Nishikimi M. 1997. Identification of S100b protein as copper-binding protein and its suppression of copper-induced cell damage. Journal of Biological Chemistry 272, 23037–23041.
Nylander M, Svensson J, Palva ET, Welin BV. 2001. Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana. Plant Molecular Biology 45, 263–279.[CrossRef][Web of Science][Medline]
Puhakainen T, Hess MW, Makela P, Svensson J, Heino P, Palva ET. 2004. Overexpression of multiple dehydrin genes enhances tolerance to freezing stress in Arabidopsis. Plant Molecular Biology 54, 743–753.[CrossRef][Web of Science][Medline]
Richard S, Morency MJ, Drevet C, Jouanin L, Seguin A. 2000. Isolation and characterization of a dehydrin gene from white spruce induced upon wounding, drought and cold stresses. Plant Molecular Biology 43, 1–10.[CrossRef][Web of Science][Medline]
Roosemont JL. 1978. Reaction of histidine residues in proteins with diethylpyrocarbonate: differential molar absorptivities and reactivities. Analytical Biochemistry 88, 314–320.[CrossRef][Web of Science][Medline]
Seki M, Narusaka M, Ishida J, et al. 2002. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. The Plant Journal 31, 279–292.[CrossRef][Web of Science][Medline]
Shen B, Jensen RG, Bohnert HJ. 1997. Mannitol protects against oxidation by hydroxyl radicals. Plant Physiology 115, 527–532.[Abstract]
Soriani M, Pietraforte D, Minetti M. 1994. Antioxidant potential of anaerobic human plasma: role of serum albumin and thiols as scavengers of carbon radicals. Archives of Biochemistry and Biophysics 312, 180–188.[CrossRef][Web of Science][Medline]
Soulages JL, Kim K, Arrese EL, Walters C, Cushman JC. 2003. Conformation of a group 2 late embryogenesis abundant protein from soybean. Evidence of poly (L-proline)-type II structure. Plant Physiology 131, 963–975.
Suzuki K, Hiroaki H, Kohda D, Nakamura H, Tanaka T. 1998. Metal ion induced self-assembly of a designed peptide into a triple-stranded -helical bundle: a novel metal binding site in the hydrophobic core. Journal of the American Chemical Society 120, 13008–13015.[CrossRef]
Svensson J, Ismail AM, Palva ET, Close TJ. 2002. Dehydrins. In: Storey KB, Storey JM, ed. Sensing, signaling and cell adaptation. Amsterdam: Elsevier, 155–171.
Svensson J, Palva ET, Welin B. 2000. Purification of recombinant Arabidopsis thaliana dehydrins by metal ion affinity chromatography. Protein Expression and Purification 20, 169–178.[CrossRef][Web of Science][Medline]
Tamura H, Goto K, Yotsuyanagi T, Nagayama M. 1974. Spectrophotometric determination of iron(II) with 1,10-phenanthroline in the presence of large amounts of iron(III). Talanta 21, 314–318.[CrossRef]
Ueda EKM, Gout PW, Morganti L. 2003. Current and prospective applications of metal ion-protein binding. Journal of Chromatography 988, 1–23.[CrossRef][Web of Science][Medline]
Vasak M, Hasler DW. 2000. Metallothioneins: new functional and structural insights. Current Opinion in Chemical Biology 4, 177–183.[CrossRef][Web of Science][Medline]
Viles JH, Cohen FE, Prusiner SB, Goodin DB, Wright PE, Dyson HJ. 1999. Copper binding to the prion protein: structural implications of four identical cooperative binding sites. Proceedings of the National Academy of Sciences, USA 96, 2042–2047.
Wisniewski M, Webb R, Balsamo R, Close TJ, Yu XM, Griffith M. 1999. Purification, immunolocalization, cryoprotective, and antifreeze activity of PCA60: a dehydrin from peach (Prunus persica). Physiologia Plantarum 105, 600–608.[CrossRef]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M.-C. Koag, S. Wilkens, R. D. Fenton, J. Resnik, E. Vo, and T. J. Close The K-Segment of Maize DHN1 Mediates Binding to Anionic Phospholipid Vesicles and Concomitant Structural Changes Plant Physiology, July 1, 2009; 150(3): 1503 - 1514. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Battaglia, Y. Olvera-Carrillo, A. Garciarrubio, F. Campos, and A. A. Covarrubias The Enigmatic LEA Proteins and Other Hydrophilins Plant Physiology, September 1, 2008; 148(1): 6 - 24. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kawachi, Y. Kobae, T. Mimura, and M. Maeshima Deletion of a Histidine-rich Loop of AtMTP1, a Vacuolar Zn2+/H+ Antiporter of Arabidopsis thaliana, Stimulates the Transport Activity J. Biol. Chem., March 28, 2008; 283(13): 8374 - 8383. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Liu and M. C. Mehdy A Nonclassical Arabinogalactan Protein Gene Highly Expressed in Vascular Tissues, AGP31, Is Transcriptionally Repressed by Methyl Jasmonic Acid in Arabidopsis Plant Physiology, November 1, 2007; 145(3): 863 - 874. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||