Tài liệu Báo cáo Y học: Systematic search for zinc-binding proteins in Escherichia coli potx

Systematic search for zinc-binding proteins in Escherichia coli Akira Katayama 1,2 , Atsuko Tsujii 2 , Akira Wada 3 , Takeshi Nishino 2 and Akira Ishihama 1 National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka, Japan; 2 Nippon Medical School, Department of Biochemistry and Molecular Biology, Bunkyo-ku, Tokyo, Japan; 3 Osaka Medical School, Department of Physics, Takatsuki, Osaka, Japan AsystematicsearchforEscherichia coli proteins with the zinc-binding activity was performed using the assay of radioactive Zn(II) binding to total E. coli proteins fraction- ated by two methods of two-dimensional gel electrophoresis. A total of 30–40 radioactive spots were identified, of which 14 have been assigned from N-terminal sequencing. In addition to five known zinc-binding proteins, nine zinc- binding proteins were newly identified including: acetate kinase (AckA), DnaK, serine hydroxymethyltransferase (GlyA), transketolase isozymes (TktA/TktB), translation elongation factor Ts (Tsf), ribosomal proteins L2 (RplB), L13 (RplM) and one of S15 (RpsO), S16 (RpsP) or S17 (RpsQ). Together with about 20 known zinc-binding pro- teins, the total number of zinc-binding proteins in E. coli increased up to more than 30 species (or more than 3% of about 1000 proteins expressed under laboratory culture conditions). The specificity and affinity of zinc-binding were analysed for some of the zinc-binding proteins. Keywords: zinc-binding protein; Escherichia coli;proteome; two-dimensional gel electrophoresis. Zinc is an essential trace element, but virtually nontoxic, in contrast to iron, copper and mercury. Over 300 enzymes or proteins have been identified that require zinc for function [1,2]. Physical and chemical properties of zinc, such as its stable association with proteins and its co-ordination flexibility, make it highly adaptable to meeting the needs of proteins and enzymes that carry out diverse biological functions [3]. In zinc-containing enzymes or proteins, zinc has two major functions, i.e. catalytic and structural. The catalytic role specifies that zinc participates directly in enzyme catalysis, while structural zinc atoms are required for stabilization of proteins by supporting their folding and oligomerization. Zinc is therefore not simply the cofactor for enzyme catalytic functions but also the structural factor for folding of domains involved in protein–protein and protein–DNA interactions. A large majority of the zinc-containing enzymes have a single zinc site consisting of a combination of specific amino-acid residues such as Cys, His, Asp and Glu, and a solvent water molecule completing the co-ordination sphere [3]. After the finding of a number of zinc-containing DNA- binding proteins in higher eukaryotes, many different types of the zinc-binding motif have been identified, including those tetrahedrally co-ordinated to imidazole nitrogen atoms from His and thiol groups from Cys [2]. The functions of protein-bound zinc are beginning to catch up with the increasing number of zinc-containing proteins. Up to the present time, the structural and functional roles of zinc have been analysed in detail with zinc-containing proteins from higher eukaryotes, but little is known about the zinc-binding proteins in prokaryotes. Ros homologues that exit in plant-associated agrobacteria are the only bacterial proteins with the typical C2H2-type zinc-finger motif [4]. Various types of zinc-containing protein with the zinc finger or zinc cluster exist in yeast, which, however, lacks proteins with the hormone receptor-type zinc-binding motif. This type of zinc-binding proteins appears in Caenorhabditis elegans [5] and the number of this type of zinc-binding proteins increases in higher animals. The aim of this study is to identify as many zinc-binding proteins as possible in the model prokaryote, Escherichia coli. For a systematic and experimental detection of zinc- binding proteins, we employed a conventional method of radioactive zinc blotting with whole cell extracts fraction- ated by two methods of two-dimensional gel electrophor- esis, the widely used O’Farrell system [6] and the newly developed radical-free and highly reducing (RFHR) method [7]. Results indicate that most of the newly identified bacterial zinc-binding proteins do not contain the known zinc-binding motifs, most of which have been identified in higher eukaryotes. This report also describes the affinity and specificity of zinc binding for some of the E. coli zinc- binding proteins. MATERIALS AND METHODS Preparation of cell extracts E. coli W3110 A-type [8] was grown in Luria–Bertani medium until late exponential phase (D 600 ¼ 0.8 ). Cells were harvested by centrifugation and cell extracts were prepared according to Iwakura et al. [9]. In brief, cells were suspended in lysis buffer (50 m M Tris/HCl, pH 8.0 at 4 °C, 1m M EDTA) and lysozyme and phenylmethanesulfonyl fluoride were added to the final concentration of 1 mgÆmL )1 and 1 m M , respectively. After incubation for 10 min at Correspondence to A. Ishihama, National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka 411-8540, Japan. Fax: + 81 559 81 6746, Tel.: + 81 559 81 6741, E-mail: aishiham@lab.nig.ac.jp Abbreviations: PVDF, poly(vinylidene difluoride); RFHR, radical-free and highly reducing. (Received 16 July 2001, revised 28 February 2002, accepted 22 March 2002) Eur. J. Biochem. 269, 2403–2413 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02900.x 30 °C, Triton X-100 (Sigma), MgCl 2 and DNase I (Sigma) were added to the final concentration of 2% (v/v), 10 m M and 16.7 UÆmL )1 , respectively. After removal of insoluble materials (unlysed cells and cell wall aggregates) by centrif- ugation at 10 000 g for 10 min at 4 °C, the supernatant fraction was used as the whole cell extract. The supernatant fraction was prepared after removal of ribosomes by centrifugation for 1 h at 100 000 g at 4 °C. Protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad). Total acid-soluble proteins were prepared from the whole cell lysate by adding 2 vol. of acetic acid at 4 °C [10]. One hour after addition of acetic acid, the acid-treated extract was centrifuged for 10 min at 10 000 g at 4 °Ctoremove acid-precipitated proteins. Two dimensional gel electrophoresis Two methods of two-dimensional gel electrophoresis system were employed throughout this study. In the O’Farrell method [6], proteins were separated according to isoelectric point by isoelectric focusing in the first dimension and then according to molecular size after denaturation with SDS in the second dimension. In this series of experiments, we employed the Pharmacia system and apparatus. In brief, the 18-cm Dry Strips, pH 4–7 (Pharmacia), were rehydrated for 12 h with 0.5 mL of rehydration solution [8 M urea, 0.5% (v/v) Triton X-100, 0.075% (w/v) dithiothreitol, 0.5% (v/v) pH 4–7 IPG buffer (Pharmacia)] containing appropriate amounts of the whole cell extract or the soluble protein fraction. First-dimension isoelectric focusing was conducted for 35 000 Vh using the Multiphor II system (Pharmacia). The first-dimension Strips were soaked first in 10 mL of equilibration buffer A [50 m M Tris/HCl, pH 6.8 at 4 °C, 30% (v/v) glycerol, 0.5% (w/v) dithiothreitol, 1% (w/v) SDS, and 6 M urea] and then in 10 mL of buffer B [50 m M Tris/HCl, pH 6.8 at 4 °C, 30% (v/v) glycerol, 4.5% isoacetamide, 1% (w/v) SDS, and 6 M urea]. The gels were then subjected to electrophoresis on the second-dimension polyacrylamide gel (Excel Gel XL-SDS 12–14). On the other hand, the RFHR method of two-dimen- sional gel electrophoresis separates proteins in the presence of urea but without using SDS and under radical-free and highly reducing conditions to avoid oxidation of proteins during electrophoresis [7]. The two-dimensional electro- phoresis was performed essentially according to the pub- lished procedure [7] after slight modification as described by Wada et al. [11] using the modified electrophoresis appar- atus (Nippon Eido Co., Japan). Blotting of radioactive zinc Proteins in the second dimension gels were blotted onto poly(vinylidene difluoride) (PVDF) membranes (Nippon Genetics, Japan) using a semidry blotting apparatus (Bio- Rad). The protein-blotted membranes were subjected to binding assay of radioactive zinc essentially according to the method of Mazen et al. [12]. In brief, the protein-blotted membranes were soaked with buffer A (10 m M Tris/HCl, pH 7.5 at 4 °C) for 1 h at room temperature, and then incubated in buffer B (10 m M Tris/HCl, pH 7.5 at 4 °C, 0.1 M KCl) containing 0.2 lCiÆmL )165 ZnCl 2 (specific activ- ity, 1.84 mCiÆmg )1 ; NEN Life Science Products, Inc.) for 15 min. After washing for 15 min in buffer B, the membranes were exposed to imaging plates and the plates were analyzed with a BAS-2000 imaging analyzer (Fuji Film Co., Japan). Construction of expression plasmids for zinc-binding proteins The genes coding for zinc-binding proteins were amplified by PCR using the whole genome DNA of E. coli W3110 (A) as template. The forward primers used included the NdeI site sequence, while the reverse primers included the EcoRI or BamHI site sequences. The PCR products were directly inserted between NdeI and either EcoRI or BamHI sites of an expression vector pGET1 [13]. The reverse primer also contained the hexahistidine (His) tag sequence; expression of the target proteins was as fusions with the His tag. Restriction enzymes used were purchased from Takara Shuzo, Japan. Expression and purification of zinc-binding proteins The expression plasmids for zinc-binding proteins were transformed into E. coli BL21(DE3) and JM109 (DE3)- pLysS. The transformed cells were grown in Luria–Bertani medium containing ampicillin (200 lgÆmL )1 ) and incubated at 37 °C with shaking. The expression level of zinc-binding proteins was compared between two recipient strains by adding 0.5 m M isopropyl thio-b- D -galactoside. For expres- sion of Def, DnaJ and RplM, E. coli JM109 transformants were used for large-scale induction, while AckA, Fur, MerR, Ppa and RpoA were expressed in E. coli BL21. For purification of zinc-binding proteins, cells were lysed in lysis buffer containing 0.2 mgÆmL )1 lysozyme, 0.2 m M phenylmethanesulfonyl fluoride and 1 m M dithiothreitol, followed by sonication. The cell lysates were centrifuged for 2 h at 100 000 g at 4 °C, and the supernatant was directly subjected to affinity chromatograpy on Ni 2+ -nitrilotriacetic acid agarose columns (Qiagen) previously equilibrated with lysis buffer. After washing with lysis buffer containing 3 m M imidazole, column-bound proteins were eluted with elution buffer containing 200 m M imidazole. Peak fractions of each zinc-binding protein were pooled and after dialysis against storage buffer [10 m M Tris/HCl, pH 8.0 at 4 °C, 10 m M MgCl 2 ,0.1m M EDTA, 1 m M dithiothreitol, 0.2 M KCl and 50% (v/v) glycerol], stored at )80 °C until use. Protein concentration was determined with a Bio-Rad protein assay kit, while the content of each zinc-binding protein was measured after separation by SDS/PAGE followed by staining with Commassie Brilliant Blue R250. The stained band intensity was measured with LAS-1000 image analyser (Fuji Film Co., Japan). Sp1 was kindly donated by Y. Sugiura (Kyoto University, Japan). RESULTS Zinc-binding assay of total E. coli proteins fractionated by two dimensional gel electrophoresis Whole cell extracts (300–400 lg proteins) of exponential- phase E. coli W3110 type A [8] were separated by two- dimensional gel electrophoresis by the widely used O’Farrell method [6]. Proteins in gels were blotted onto PVDF membranes and then subjected to the binding assay of 2404 A. Katayama et al. (Eur. J. Biochem. 269) Ó FEBS 2002 65 ZnCl 2 . Under the experimental conditions employed, about 300–400 protein spots were identified after staining with Coomassie Brilliant Blue (Fig. 1A), of which 20–30 spots were identified to be labelled after exposure to radioactive Zn(II) (Fig. 1B), suggesting that about 5–10% protein species have the Zn(II)-binding activity. The Zn(II)-binding assay was then performed with the soluble fraction after removal of the group of abundant proteins such as membrane proteins, ribosomal proteins and nucleoid-associated DNA-binding proteins (or nucleoid proteins). The overall pattern of Zn(II) binding was essentially the same with that obtained using whole cell extracts (compare Fig. 1B,D). Some Zn(II)-binding spots detected with the whole cell extracts disappeared, and instead some new radioactive spots were detected because of the increase in relative levels for those proteins in the soluble protein fraction. Because the intensity of Zn(II) binding and the staining intensity with CBB do not correlate and because the pattern of radioactive Zn(II) binding differs between two different preparations of the cell extract, we concluded that the filter binding assay herein employed allows the detection of at least a group of proteins with Zn(II)-binding activity. It should be noted, however, that the intensity of Zn(II) radioactivity thus detected reflects both the affinity to Zn(II) and the protein concentration. To increase in the resolution of basic proteins, we also employed the RFHR method of two-dimensional gel electrophoresis [7]. In addition, artefacts arising from oxidation of proteins during electrophoresis could also be avoided by using the RFHR method. Figure 2 shows one example of the Zn(II)-binding assay for acid-soluble basic proteins separated by the RFHR method. Some of the basic proteins were newly identified as having Zn(II)-binding activity. After sequence analysis, these basic proteins were identified as specific ribosomal proteins (see below). The ribosomal proteins detected in the region of gel electro- Fig. 1. Radioactive Zn(II) binding assay of E. coli total proteins fractionated by the O’Farrell method of two-dimensional gel electrophoresis. Awhole cell extract (A and B, 400 mg) or a soluble protein fraction (C and D, 400 mg) of exponentially growing E. coli W3110 type A were fractionated by the O’Farrell method [6] of two-dimensional PAGE. After electrophoresis, proteins were blotted onto PVDF membranes followed by staining with Commassie Brilliant Blue R250 (A and C). The protein-blotted membranes were also subjected to the binding assay of radioactive Zn(II) as described in Materials and methods (B and D), and then exposed to imaging plates which were analysed with a BAS-2000 image analyser. The Zn(II)-binding proteins indicated by arrows were analysed for the N-terminal sequences. The spot numbers correspond to the gene numbers listed in Table 1. Ó FEBS 2002 Escherichia coli zinc-binding proteins (Eur. J. Biochem. 269) 2405 phoresis exist roughly in stoichiometric amounts, but the 65 Zn radioactivity was detected with only some specific ribosomal protein spots, supporting the prediction that the Zn(II)-binding activity detected by the method herein employed represents specific binding. Taken together we concluded that a total number of the Zn(II)-binding protein in E. coli ranges between 20 and 30 proteins among 300–400 major proteins expressed under the culture condition employed. Protein sequence analysis for the isolated Zn(II)-binding proteins In order to identify the nature of proteins showing Zn(II)- binding activity, we isolated the major Zn(II)-binding proteins from PVDF membranes and subjected them to N-terminal microsequencing. Up to the present time, we succeeded in identifying the N-terminal sequences for 14 protein spots (Table 1; and also see Figs 1B, 1D and 2B for the location of these proteins on the two-dimensional gel patterns), among which four spots included two (AhpC/ Ppa, AtpA/LpdA, and TktA/TktB) or three (RpsO/RpsP/ RpsQ) protein sequences, indicating these two or three proteins migrated to the same positions on two-dimensional gel electrophoresis. In the case of AhpC/Ppa spot, Ppa was identified to be the Zn(II)-binding component, because the isolated inorganic pyrophosphatase (Ppa) exhibited the Zn(II)-binding activity (see below), in agreement with the previous observations [14–16]. Lipoamide dehydrogenase (LpdA) [17], fructose-1,6-bisphosphatase aldolase (Fba) [18,19], phosphotransacetylase (Pta) [20] and RNA poly- merase a subunit (RpoA) [21] have all been shown to contain or bind zinc in its isolated state. All these observations indicate that the method employed in this study is useful for the identification of yet unidentified Zn(II)-binding proteins. Because the intracellular concen- trations of Zn(II)-binding proteins thus identified are, however, different in the cell extracts analysed, the different intensity of radioactivity for these spots does not necessarily represent the relative affinity of Zn(II)-binding. Fig. 2. Radioactive Zn(II) binding assay of E. coli acid-soluble proteins fractionated by the RFHR method of two-dimensional gel electrophoresis. Acid-soluble proteins (380 mg), prepared from the whole cell lysate of exponentially growing E. coli W3110 type-A, were fractionated by the RFHR method [7] of two-dimensional PAGE. After electrophoresis, proteins were blotted onto PVDF membranes followed by staining with Commassie Brilliant Blue R250 (A). The locations of low-molecular-mass ribosomal proteins are indicated. The protein-blotted membrane was also subjected to the binding assay of radioactive Zn(II) as described in Materials and methods (B), followed by exposure to an imaging plate, which was then analysed with a BAS-2000 image analyser (Fuji, Japan). The Zn(II)-binding proteins indicated by arrows were analysed for the N-terminal sequences. The spot numbers correspond to the gene numbers listed in Table 1. Table 1. Zinc binding proteins. Protein spots with zinc-binding activity were cut out from PVDF membranes and immediately subjected to N-terminal microsequencing. From the sequence results, the genes coding for these proteins were identified. Protein spots, AhpC/Ppa, AtpA/LpdA, RpsO/RpsP/RpsQ and TktA/Tkt, were found to contain multiple proteins, among which Ppa and LpdA were identified to have the zinc-binding activity. Asterisks indicate the previouslyidentified zinc-containing proteins in E. coli (see Table 2). Spot Gene product Function 1 AckA Acetate kinase 2 DnaK Hsp70 chaperone 3 Fba* Fructose 1,6-bisphosphatase aldolase 4 GlyA Serine hydroxymethyltransferase 5 LpdA* (AtpA) Lipoamide dehydrogenase (F1 ATP synthase a subunit) 6 Ppa* (AhpC) Inorganic pyrophosphatase (alkyl hydroperoxide reductase C22) 7 Pta* Phosphotransacetylase 8 RpoA* RNA polymerase a subunit 9 RplB 50S ribosomal protein L2 10 RplM 50S ribosomal protein L13 11 RpsB 30S ribosomal protein S2 12 RpsO/RpsP/RpsQ 30S ribosomal protein S15, S16 or S17 13 TktA/TktB Transketolase isozymes 14 Tsf Translation elongation factor Ts 2406 A. Katayama et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Confirmation of the Zn(II)-binding activity using purified proteins To confirm the Zn(II)-binding activity and to estimate the Zn(II)-binding affinity for the newly identified Zn(II)- binding proteins, we next cloned the genes encoding some of the newly identified Zn(II)-binding proteins into expres- sion vectors, over-expressed His 6 -tagged proteins in trans- formed E. coli cells, and affinity-purified the His 6 -tagged proteins to apparent homogeneity. Equal amounts of the purified Zn(II)-binding proteins were subjected to the Zn(II)-binding assay. Figure 3 shows the 65 Zn(II)-binding activity for two proteins, RplM (ribosomal protein L13) and RpoA (RNA polymerase a subunit), in parallel with four known heavy metal-binding proteins, Zn(II)-binding Def (peptide deformylase) [22,23], Hg(II)-binding MerR (mer- cury export regulation protein) with Zn(II)-binding activity [24,25] and Fe(II)-binding Zn(II)-containing Fur (ferric uptake regulation protein) [26,27] and Zn(II)-binding BSA (bovine serum albumin) [28], and one control protein, RpoD (RNA polymerase r subunit) with no known activity of Zn(II)-binding. 65 Zn(II) binding was detected even at low protein concentrations for Def, MerR, Fur and RpoA (Fig. 3C), and in addition, for RplM and BSA at high protein concentrations (Fig. 3D). In addition to RplM and RpoA, Zn(II)-binding activity was confirmed for other three purified proteins, AckA (acetate kinase), Ppa (inor- ganic pyrophosphatase) and DnaJ (see below). Here we detected a low level of Zn(II) binding for RpoD, the RNA polymerase r 70 subunit (see also below). At present, however, it is not clear whether this represents a background activity of nonspecific Zn(II) binding or RpoD carries a weak but specific Zn(II)-binding site. The test proteins were isolated as fusion forms with His 6 - tag added at the C-terminus. The possible influence of the His 6 tag on the Zn(II)-binding activity was then tested. The His 6 tag added to the test proteins was found to show a low level of the Zn(II)-binding activity, but the level of 65 Zn(II)- blotting by the His 6 tag was less than 25%, if any, of the total Zn(II)-binding activity by the Zn(II)-binding proteins examined (for examles see AckA, RpoA and Ppa, shown in Figs 6A and 8). To confirm this prediction, the Zn(II) binding assay was also performed for untagged proteins (see below). Fig. 3. Radioactive Zn(II)-binding assay of purified E. coli proteins. The newly identified Zn(II)-binding proteins, RplM (L13) and RpoA (RNA polymerase a subunit), and the previously identified Zn(II)-binding proteins, Def, MerR and Fur, were purified from over-expressed E. coli cells. Equal amounts (A and C, 20 pmol each; and B and D, 100 pmol each) of the purified proteins were analysed, together with whole cell lysate (A and C, 1 lg; B and D, 5 lg), BSA (A and C, 1 lg; B and D, 5 lg), RNA polymerase (A and C, 1 lg; B and D, 5 lg) and RpoD (RNA polymerase r subunit) (A and C, 1 lg; B and D, 5 lg), by SDS/PAGE. After electrophoresis, the proteins were blotted onto PVDF membranes followed by staining with Commassie Brilliant Blue R250 (A and B). The protein-blotted membranes were also subjected to the binding assay of radioactive Zn(II) as described in Materials and methods (C and D), followed by exposure to an imaging plate which was analysed with a BAS-2000 image analyser. CH represents the hexahistidine (His) tag added at the C-terminus of each protein. RPase core indicates the RNA polymerase core enzyme with the subunit composition a 2 bb¢. Ó FEBS 2002 Escherichia coli zinc-binding proteins (Eur. J. Biochem. 269) 2407 Affinity of Zn(II) binding by the purified Zn(II)-binding proteins Using the purified proteins, we then measured the Zn(II)- binding affinity. For this purpose, the level of protein-bound 65 Zn(II) was measured using the same amounts of the newly identified Zn(II)-binding proteins, AckA and Ppa, and the known metal-binding proteins, Def, MerR and Fur, in the presence of a fixed amount of radioactive Zn(II) and increasing concentrations of nonradioactive Zn(II). The level of Zn(II) binding to all the test proteins increased concomitantly with the increase of Zn(II) concentration [note the decrease in specific radioactivity of 65 Zn(II)] (Fig. 4). The affinity of Zn(II) binding by both AckA and Ppa was estimated to be within the same range of those of the reference proteins, Def, MerR and Fur. Specificity of Zn(II) binding by the whole cell extracts To examine the specificity of Zn(II) binding for the known and novel E. coli Zn(II)-binding proteins, we performed the competitive inhibition assay of 65 Zn(II) binding with other metals. The pattern of radioactive Zn(II) binding by the whole cell extract (Fig. 5B) was essentially the same with that observed in Fig. 1. By the addition of 100 l M nonradioactive Zn(II), the concentration giving more than 90% inhibition (see Fig. 4), the level of radioactive Zn(II) binding decreased to below 10% (Fig. 5C). The level of radioactive Zn(II) binding, however, remained essentially at the same levels by the addition of 100 l M Mg(II) (Fig. 5D), Ni(II) (Fig. 5E) and Cd(II) (Fig. 5F). The results indicate that the activity of Zn(II) binding detected by the conven- tional filter binding assay of radioactive Zn(II) represents the specific binding activity to Zn(II), but is not attributable to nonspecific metal binding. Specificity of Zn(II) binding by the purified Zn(II)-binding proteins The specificity of Zn(II) binding was also examined for the purified Zn(II)-binding proteins. The binding of radioactive Zn(II) to AckA, Def, DnaJ, Fur, MerR, Ppa, RplM, and RpoA was tested in the presence of 100 l M nonradioactive ZnCl 2 ,MgCl 2 or FeCl 3 . The amount of radioactive Zn(II) binding decreased by the addition of nonradioactive Zn(II) (Fig. 6B) and the reduction level was apparently the same between the test proteins. In contrast, the Zn(II) binding activity by these proteins was not affected by the addition of Mg(II) (Fig. 6C) and Fe(III) (Fig. 6D). Although the Fur protein has a high affinity to Fe(III), the binding of radioactive Zn(II) to Fur was not interfered with the addition of Fe(III) (Fig. 6D), being consistent with the finding that the site of zinc binding is different from the iron- binding site [27]. The Zn(II) binding activity of AckA, Def, DnaJ, Fur, MerR, Ppa, and RplM was also tested in the presence of Ca(II), Cu(II) and Cd(II) (Fig. 7), but none of these metals affected the binding of radioactive Zn(II) to the test proteins. Taken the results of all these competition assays together we concluded that the observed Zn(II)-binding activity by the test proteins represents the specific binding of Zn(II). Fig. 4. Dose-dependent binding of Zn(II) to purified E. coli zinc-binding proteins. The newly identified E. coli Zn(II)-binding proteins, AckA and Ppa, were analysed, together with the known Zn(II)-binding proteins, Def, MerR, Fur and Sp1, for radioactive Zn(II) binding in the presence of indicated concentrations of nonradioactive ZnCl 2 . Proteins analysed were: lane 1, AckA(CH) + Ppa(CH) + MerR(CH) + Sp1; and lane 2, Def(CH) + Fur(CH). After electrophoresis, the proteins were blotted onto PVDF membranes and the protein-blotted membranes were subjected to the binding assay of 65 ZnCl 2 (0.2 lCiÆmL )1 ) in the presence of indicated concentrations of nonradioactive ZnCl 2 , followed by exposure to imaging plates which were then analysed with a BAS-2000 image analyser. 2408 A. Katayama et al. (Eur. J. Biochem. 269) Ó FEBS 2002 To exclude the influence of His-tag on the activity and specificity of Zn(II) binding, we checked the specificity of Zn(II) binding using purified proteins without the His-tag. The Zn(II)-binding specificity was examined for untagged EF-Tu (Tuf), EF-Ts (Tsf), RpoA, Ppa and Sp1. The radioactive Zn(II) binding was competed by nonradioactive Zn(II) but not by Ca(II), Cd(II), Mg(II) and Ni(II) (Fig. 8A,B). Again a low level of Zn(II)-binding activity was detected for RpoD when analysed using a large amount of protein. Taken together we conclude that the activity of Zn(II) binding herein detected is not due to the His 6 tag but the E. coli proteins (and Sp1) and that the binding is specific to Zn(II) and does not occur for other divalent metals. DISCUSSION Physiological roles of the protein-bound zinc in enzyme catalytic functions and formation of protein functional domains, in particular those involved in protein–protein and protein–nucleic acid interactions, have been studied in Fig. 5. Competition of radioactive Zn(II) binding to E. coli proteins by nonradioactive metals. About 400 mg of the soluble fraction of whole cell extract of exponentially growing E. coli W3110 type A [8] was fractionated by the O’Farrell method [6] of two-dimensional PAGE. After electrophoresis, proteins were blotted onto PVDF membranes followed by staining with Commassie Brilliant Blue R250 (A). The protein-blotted membranes were also subjected to the binding assay of radioactive Zn(II) as described in Fig. 1B in the absence (B) or presence of 100 l M ZnCl 2 (C), MgCl 2 (D), NiCl 2 (E) or CdCl 2 (F). The filters were treated as described in Fig. 4. Fig. 6. Competition of radioactive Zn(II) binding by nonradioactive metals, Zn(II), Mg(II) and Fe(III), to purified E. coli zinc-binding proteins. Mixtures of the purified Zn(II)-binding proteins were subjected to radioactive Zn(II)-binding assay in the absence (A) or presence of 100 l M nonradioactive Mg(II) (B), Zn(II) (C) or Fe(III) (D). Proteins analysed were: Lane 1, AckA; lane 2, AckA(CH) + Ppa(CH) + Sp1; lane 3, Ppa; lane 4, RpoA(CH) + RplM(CH); lane 5, RpoA + MerR(CH); lane 6, Fur(CH); lane 7, Def(CH); lane 8, DnaJ(CH). After SDS/PAGE, gels were treated as described in Fig. 4. Ó FEBS 2002 Escherichia coli zinc-binding proteins (Eur. J. Biochem. 269) 2409 details for zinc-containing metalloproteins from higher animals (reviewed in [3]). In contrast, relatively little is known about the roles of zinc in function and structure of proteins from prokaryotes. The total number of known zinc-containing E. coli protein species that have been experimentally examined to date is not more than 20 (Table 2). Here we performed, for the first time in E. coli molecular genetics, a systematic search for E. coli proteins Fig. 7. Competition of radioactive Zn(II) binding to purified E. coli zinc-binding proteins by nonradioactive metals, Ca(II), Cu(II) and Cd(II). Mixtures of the purified Zn(II)-binding proteins (100 pmol each) were subjected to the Zn(II)-binding assay ( 65 ZnCl 2 ,0.2lCiÆmL )1 )intheabsenceor presence of 100 l M nonradioactive Zn(II), Ca(II), Cu(II) or Cd(II). Proteins analysed were: lane 1, DnaJ(CH) + Sp1; lane 2, MerR(CH); lane 3, RpoA + Def(CH) + Fur(CH); and lane 4, AckA(CH) + Ppa(CH) + RplM(CH). After SDS/PAGE, gels were treated as described in Fig. 4. Fig. 8. Competition of radioactive Zn(II) binding to purified zing-binding proteins without His-tag by nonradioactive metals. Purified zinc-binding proteins without His-tag were analysed for radioactive Zn(II) binding in the presence or absence of nonradioactive metals as indicated on bottom of each gel lane. (A) Lane 1, EF-Tu + EF-Ts; lane 2, RpoD + RpoA; lane 3, AckA + Ppa + Sp1. (B) Lanes 1–6, AckA + Ppa. 2410 A. Katayama et al. (Eur. J. Biochem. 269) Ó FEBS 2002 with the Zn(II)-binding activity, by using the conventional radioactive Zn(II)-binding assay onto filter-bound proteins after separation of total proteins by two-dimensional gel electrophoresis. About 20–30 proteins have been identified to have the binding activity of 65 Zn(II) (see Figs 1 and 2). After N-terminal sequencing, the nature of proteins has been identified for 14 species, including five known zinc- containing proteins, Fba (fructose-1,6-bisphosphatase aldo- lase), LpdA (lipoamide dehydrogenase), Ppa (inroganic pyrophosphatase), Pta (phosphotransacetylase) and RpoA (RNA polymerase a subunit) (compare Tables 1 and 2). Because the level of radioactive Zn(II) association did not correlate with the amount of protein, the Zn(II) binding capability detected by the method herein employed was considered to represent the intrinsic activity of Zn(II) binding. Thus the Zn(II)-binding proteins herewith identi- fied may require Zn(II) as an intrinsic cofactor for either the formation of native conformations or the expression of their intrinsic functions. The success of detection of specific Zn(II)-binding proteins agrees with the prediction that the formation of Zn(II)-binding site includes short stretches of the protein sequence that can be easily refolded after denaturation during electrophoretic separation of proteins. The E. coli proteins detected in this study must belong to a group of proteins with similar affinity to Zn(II). In fact, the binding affinity of Zn(II) as measured by competition assay (see Fig. 4) was similar between this group of proteins. Conse- quently It can not be excluded that Zn(II)-binding motifs, which require longer stretches of the protein and can not be refolded after one cycle of denaturation-renaturation treat- ment, could not be detected by the method herein employed. Under laboratory culture conditions, E. coli expresses at most 1000 genes out of 4000 ORFs on the genome, as detected by two-dimensional gel electrophoresis. In this study, we identified 20–30 Zn(II)-binding proteins among 300–400 E. coli proteins (or 5–10% of total open reading frames). If the relative amount of Zn(II)-binding proteins in terms of total protein species is the same for other E. coli proteins, the total number of zinc-binding proteins in E. coli could be about 200–400 (or 5–10% of a total of 4000 open reading frames on the E. coli genome). Most of the Zn(II)- binding proteins herein detected do not contain any of the known zinc-binding motifs, which were identified in zinc-containing proteins from higher eukaryotes. After a computational search for Zn(II)-binding E. coli proteins, however, we identified only a total of about 30 proteins (N. Fujita & A. Ishihama, unpublished results). These findings suggest that most of the E. coli Zn(II)-binding proteins is composed of unidentified Zn(II)-binding motifs that are different from the known eukaryote-type Zn(II)- binding motifs. In addition to the known zinc-containing proteins, we obtained in this study experimental evidence showing Zn(II) binding for some E. coli proteins, including acetate kinase (AckA) that is known to require the metal for expression of the catalytic function [29], DnaK that is considered to carry zinc as in the case of DnaJ [30,31], and serine hydroxymeth- yltransferase (GlyA), which requires a metal as a cofactor for its enzyme function. Previously, none of the ribosomal proteins in E. coli have been identified as having binding activity for Zn(II). Here we identified for the first time that Zn(II) can bind to at least four ribosomal proteins, L2 (RplB), L13 (RplM), S2 (RpsB) and one (or two) of S15 (RpsO), S16 (RpsP) or S17 (RpsQ). The possible involve- ment of zinc in either the assembly of these ribosomal proteins into ribosomes or the expression of intrinsic functions of these ribosomal proteins is to be examined. The secX gene product of E. coli is now recognized as the smallest subunit L36 of 50S large ribosomal subparticle [32]. From the solution structure by NMR, the 37-amino-acid Thermus thermophilus L36 protein was found to contain zinc at a zinc-ribbon-like fold [33] even though the function of L36 is yet unidentified. The pattern of three Cys residues and a following His residue in the bacterial L36 protein, however, does not match that of any other known zinc-finger protein. In the two-dimensional gel electrophoresis conditions employed (Fig. 2), however, L36 migrated outside the gel space that was subjected to Zn(II)-binding assay. ACKNOWLEDGEMENTS We thank Katsunori Yata (National Institute of Genetics, RI Centre) for expression and purification of Fur and MerR proteins, and Yukio Sugiura (Kyoto University) for the gift of Sp1. This work was supported by Grants-in-Aid from the Ministry of Education, Science, Culture and Sports of Japan, and by CREST fund from the Japan Science Corporation (JSP). REFERENCES 1. Vallee, B.L. & Auld, D.S. (1990) Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29, 5647–5659. 2. Coleman, J.E. (1992) Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu. Rev. Bio- chem. 61, 897–946. 3. Vallee, B.L. & Falchuk, K.H. (1993) The biochemical basis of zinc physiology. Physiol. Rev. 73, 79–118. 4. Bouhouche, N., Syvanen, M. & Kado, C.I. (2000) The origin of prokaryotic C2H2 zinc finger regulators. Trends Microbiol. 8, 77–81. Table 2. Zinc-binding proteins in Escherichia coli. 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In addition to five known zinc-binding proteins, nine zinc- binding proteins were newly identified including: acetate kinase (AckA),. of zinc-binding proteins The expression plasmids for zinc-binding proteins were transformed into E. coli BL21(DE3) and JM109 (DE3)- pLysS. The transformed