Systematic search for zinc-binding proteins in Escherichia coliAkira Katayama1,2, Atsuko Tsujii2, Akira Wada3, Takeshi Nishino2and Akira Ishihama 1 National Institute of Genetics, Depart
Trang 1Systematic search for zinc-binding proteins in Escherichia coli
Akira Katayama1,2, Atsuko Tsujii2, Akira Wada3, Takeshi Nishino2and 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
A systematic search for Escherichia 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 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
M A T E R I A L S A N D M E T H O D S
Preparation of cell extracts
E coli W3110 A-type [8] was grown in Luria–Bertani medium until late exponential phase (D600 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 mMTris/HCl, pH 8.0 at 4C,
1 mM EDTA) and lysozyme and phenylmethanesulfonyl fluoride were added to the final concentration of 1 mgÆmL)1 and 1 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)
Trang 230C, Triton X-100 (Sigma), MgCl2and DNase I (Sigma)
were added to the final concentration of 2% (v/v), 10 mM
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 4C, 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 4C.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 4C [10].One
hour after addition of acetic acid, the acid-treated extract
was centrifuged for 10 min at 10 000 g at 4C to remove
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 [8Murea, 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 mM Tris/HCl, pH 6.8 at 4C,
30% (v/v) glycerol, 0.5% (w/v) dithiothreitol, 1% (w/v)
SDS, and 6Murea] and then in 10 mL of buffer B [50 mM
Tris/HCl, pH 6.8 at 4C, 30% (v/v) glycerol, 4.5%
isoacetamide, 1% (w/v) SDS, and 6Murea].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 mMTris/HCl,
pH 7.5 at 4C) for 1 h at room temperature, and then
incubated in buffer B (10 mM Tris/HCl, pH 7.5 at 4C,
0.1MKCl) containing 0.2 lCiÆmL)1 65ZnCl2(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 37C with shaking.The expression level of zinc-binding proteins was compared between two recipient strains by adding 0.5 mMisopropyl 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 mM phenylmethanesulfonyl fluoride and 1 mM dithiothreitol, followed by sonication.The cell lysates were centrifuged for
2 h at 100 000 g at 4C, and the supernatant was directly subjected to affinity chromatograpy on Ni2+-nitrilotriacetic acid agarose columns (Qiagen) previously equilibrated with lysis buffer.After washing with lysis buffer containing 3 mM imidazole, column-bound proteins were eluted with elution buffer containing 200 mMimidazole.Peak fractions of each zinc-binding protein were pooled and after dialysis against storage buffer [10 mM Tris/HCl, pH 8.0 at 4C, 10 mM MgCl2, 0 1 mMEDTA, 1 mMdithiothreitol, 0.2MKCl 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)
R E S U L T S
Zinc-binding assay of totalE 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
Trang 365ZnCl2.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 A whole 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.
Trang 4phoresis exist roughly in stoichiometric amounts, but the
65Zn 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)-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).
aldolase
(F1 ATP synthase a subunit)
(alkyl hydroperoxide reductase C22)
12 RpsO/RpsP/RpsQ 30S ribosomal protein S15, S16 or
S17
Trang 5Confirmation of the Zn(II)-binding activity
using purified proteins
To confirm the Zn(II)-binding activity and to estimate the
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 His6-tagged proteins in
trans-formed E coli cells, and affinity-purified the His6-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 the65Zn(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.65Zn(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 r70 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 His6 -tag added at the C-terminus.The possible influence of the His6tag on the Zn(II)-binding activity was then tested.The His6tag added to the test proteins was found to show a low level of the Zn(II)-binding activity, but the level of65 Zn(II)-blotting by the His6tag 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 bb¢.
Trang 6Affinity 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
65Zn(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 65Zn(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 of65Zn(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 lM
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 lMMg(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 lMnonradioactive ZnCl2, MgCl2or FeCl3.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 of65ZnCl 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.
Trang 7To 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 His6tag but the E coli proteins (and Sp1) and that the binding is specific
to Zn(II) and does not occur for other divalent metals
D I S C U S S I O N
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.
Trang 8details 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 (65ZnCl 2 , 0 2 lCiÆmL)1) in the absence or 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.
Trang 9with 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 of65Zn(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 thermophilusL36 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
A C K N O W L E D G E M E N T S
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).
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