Iron inhibits Escherichia coli topoisomerase I activity by targeting the first two zinc-binding sites in the C-terminal domain
Wu Wang,1 Xiaolu Su,1 Xiaobing Wang,2 Juanjuan Yang,1 Ting Zhang,1 Maofeng Wang,3 Rugen Wan,3 Guoqiang Tan,1 and Jianxin Lu1* 1
Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
2
Department of Rheumatism, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325035, China Department of Laboratory Medicine, Affiliated Dongyang Hospital of Wenzhou Medical University, Dongyang, Zhejiang 322100, China
3
Received 22 July 2014; Revised 26 August 2014; Accepted 27 August 2014 DOI: 10.1002/pro.2542 Published online 30 August 2014 proteinscience.org
Abstract: Escherichia coli DNA topoisomerase I (TopA) contains a 67 kDa N-terminal catalytic domain and a 30 kDa C-terminal zinc-binding region (ZD domain) which has three adjacent tetracysteine zinc-binding motifs. Previous studies have shown that E. coli TopA can bind both iron and zinc, and that iron binding in TopA results in failure to unwind the negatively supercoiled DNA. Here, we report that each E. coli TopA monomer binds one atom of iron via the first two zincbinding motifs in ZD domain and both the first and second zinc-binding motifs are required for iron binding in TopA. The site-directed mutagenesis studies further reveal that while the mutation of the third zinc-binding motif has very little effect on TopA’s activity, mutation of the first two zincbinding motifs in TopA greatly diminishes the topoisomerase activity in vitro and in vivo, indicating that the first two zinc-binding motifs in TopA are crucial for its function. The DNA-binding activity assay and intrinsic tryptophan fluorescence measurements show that iron binding in TopA may decrease the single-stranded (ss) DNA-binding activity of ZD domain and also change the protein structure of TopA, which subsequently modulate topoisomerase activity. Keywords: Escherichia coli topoisomerase I; iron binding; zinc-binding motif; topoisomerase activity
Introduction Abbreviations: TopA, E. coli topoisomerase I; PAR, 4-(2-pyridylazo)resorcinol; ZD, 30 kDa C-terminal zinc-binding domain in TopA. Grant sponsor: The Chinese National Natural Science Foundation Grants; Grant numbers: 31228006, 31100576; Grant sponsor: The Natural Science Foundation of Zhejiang Province Grants; Grant numbers: LY13C050003, LY12C05003, LY13B070012. *Correspondence to: Jianxin Lu; Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China. E-mail: [email protected]
C 2014 The Protein Society Published by Wiley-Blackwell. V
Escherichia coli topoisomerase I (TopA) was the first discovered type I topoisomerase.1,2 Structurally, TopA contains a 67 kDa N-terminal catalytic domain (N67 domain) and a 30 kDa C-terminal zinc-binding region (ZD domain). The N-terminal domain binds double-stranded DNA and undertakes the cleavagerejoining catalytic action, but cannot complete relaxation of the negatively supercoiled DNA.3,4 The ZD domain comprises three tandem-arranged tetra-cysteine zinc-binding motifs and a 14 kDa DNA-binding domain,5–7 and is required not only for topoisomerase activity but also for interacting with RNA
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polymerase.8,9 Although crystal structures of the E. coli TopA N-terminal domain and the covalent intermediate complex interacting with single-stranded DNA have been reported,10,11 the full-length E. coli TopA crystal structure is currently not available. In previous studies, we found that E. coli TopA is able to bind both iron and zinc in E. coli cells, and that the iron-bound TopA fails to relax the negatively supercoiled DNA in vitro,12 suggesting that the enzyme activity of TopA may be regulated by intracellular iron content. Nevertheless, since the ZD domain contains three tandem-arranged tetracysteine zinc-binding motifs, the iron-binding sites in TopA remain elusive. Here we report that each E. coli TopA monomer binds one atom of iron via the first two tetracysteine zinc-binding motifs in the ZD domain. The site-directed mutagenesis studies further show that the first two zinc-binding motifs in TopA are essential for the topoisomerase activity in vitro and in vivo. Finally, iron binding in TopA appears to dramatically decrease the ssDNA-binding activity of ZD domain and the intrinsic fluorescence intensity of tryptophan in TopA protein, suggesting that iron binding in TopA may change the protein structure of TopA and subsequently modulate enzyme activity.
Results Each TopA monomer binds one atom of iron via the first two zinc-binding motifs in the ZD domain Previous studies indicated that E. coli TopA can bind both iron and zinc.12 However, the iron-binding sites in TopA are not known. Since TopA contains three zinc-binding motifs in the ZD domain,5 we constructed three TopA mutants (ZM1-mut, ZM2-mut, and ZM3-mut) in which the first two cysteine residues in the first (ZM1), second (ZM2), and third (ZM3) zinc-binding motif were replaced with serine, respectively. The wide-type TopA and each of TopA mutant proteins were expressed in the E. coli cells grown in the M9 minimal media supplemented with ferric citrate, and purified as described previously.12 The UV–Vis absorption analyses showed that the purified wide-type TopA has two major absorption peaks at 482 and 563 nm which indicate the iron binding in the protein.12 Purified ZM3-mut protein also has a similar absorption spectrum [Fig. 1(B)] and reddish color [Fig. 1(A)] as the wide-type TopA. In contrast, purified ZM1-mut and ZM2-mut have very little or no absorption peaks at 482 and 563 nm [Fig. 1(B)] and no color [Fig. 1(A)]. The total iron content analyses further revealed that wide-type TopA and ZM3-mut protein contain around one atom of iron per protein monomer, but the iron binding in ZM1 and ZM2 mutant proteins is significantly decreased [Fig. 1(C)]. We further constructed a TopA
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mutant in which both the first and second zincbinding motifs are mutated, and found that the iron content of the ZM1/ZM2 double mutant is essentially eliminated (Fig. 1). These results suggest that each wide-type TopA monomer can only bind one iron atom via the first and the second zinc-binding motifs in the ZD domain. In parallel, the wide-type TopA and each of TopA mutant proteins were expressed in the E. coli cells grown in the M9 minimal media supplemented with zinc sulfate. The measurement of zinc content shows that each wide-type TopA molecule contains three atoms of zinc as previously reported.5 In contrast, each of the three single mutants (ZM1-mut, ZM2-mut, and ZM3-mut) has two Zn(II) bound per monomer, and there is only one bound in ZM1/ZM2 double mutant [Fig. 1(D)]. The results indicate that the coordinations of Zn(II) with zinc-binding motifs in TopA is different from that of iron binding.
Both the first and the second zinc-binding motifs are required for iron binding in TopA To further explore the iron-binding sites in TopA, we subcloned the DNA fragments that encode a truncated TopA protein with deletion of the third zincbinding motif (ZM3-del), the N-terminal domain of TopA (N67), the first zinc-binding motif (ZM1), the second zinc-binding motif (ZM2), and the peptide fragment containing the first two zinc-binding motifs (ZM11ZM2) [Fig. 2(A)]. Each of these TopA fragments was successfully expressed in the E. coli cells grown in the M9 minimal media supplemented with ferric citrate. As shown in Figure 2B and C, purified ZM3-del has the same absorption peaks at 482 and 563 nm and contains almost the same amount of iron as the wild-type TopA. The ZM1 1 ZM2 fragment of TopA is also able to bind iron, although the iron content in the ZM1 1 ZM2 fragment is less than that in the intact TopA. In contrast, the purified Nterminal domain of TopA, the ZM1 and the ZM2 fragment contain negligible amount of iron [Fig. 2(B,C)]. Taken together, the results further suggest that E. coli TopA binds iron via the first and the second zinc-binding motifs, and both ZM1 and ZM2 are required for iron binding in E. coli TopA, while the third zinc-binding motif is not involve in the iron binding.
Disruption of the first two zinc-binding motifs in TopA results in an inactive enzyme in vitro and in vivo As only the first two zinc-binding motifs in the ZD domain are involved in the iron binding in TopA, it would be interesting to explore the specific roles of these zinc-binding motifs in the enzyme activity of TopA. To eliminate the effect of iron binding on topoisomerase activity of TopA,12 TopA and the TopA
Iron Inhibits Escherichia coli Topoisomerase I Activity
Figure 1. Iron-binding activity and zinc-binding activity of E. coli TopA and TopA mutants. (A) A photograph of E. coli TopA and TopA mutant proteins (40 lM) purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. (B) UV–Vis absorption spectra of purified E. coli TopA and TopA mutants. The concentration of the proteins was about 40 lM. (C) The iron content of E. coli TopA and TopA mutant proteins purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. (D) The zinc content of E. coli TopA and TopA mutant proteins purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ZnSO4. Results were the mean values plus or minus standard deviations from three independent experiments.
variants were prepared from the E. coli cells grown in the M9 minimal medium supplemented with 50 lM ZnSO4. Purified proteins were subjected to the in vitro topoisomerase activity assay as described previously.12 Figure 3(A) shows that the topoisomerase activity of ZM1-mut or ZM2-mut is dramatically decreased, while ZM3-mut is reduced by about 50%. When both the ZM1 and ZM2 are mutated, the TopA protein has no detectable activity [Fig. 3(A)], suggesting that the first two zinc-binding motifs are essential for the topoisomerase activity in vitro. TopA and the variants were also prepared from the E. coli cells grown in M9 minimal medium supplemented with 50 lM ferric citrate. As shown in Figure 3(B), the topoisomerase activity of the ironbound wide-type TopA, ZM1-mut, ZM2-mut, and ZM3-mut proteins are all inactive, consistent with the previous results that the iron-bound TopA is not active.12
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The enzyme activity of the TopA variants was further subjected to complementation analysis. The E. coli strain RFM475 lacks the topA gene but contains compensatory mutations in gyrB necessary for viability of RFM475, so it grows normally at 37 C. However, its survival would be threatened if the cells were exposed to high (52 C) and low (30 C) temperatures or oxidative stress.13–15 To test the in vivo function of the constructed TopA variants, the wild-type TopA and TopA variants were expressed in the RFM475 strain. The E. coli strain RFM475 expressing the wild-type TopA and TopA variants were grown in LB media at 28 C. As shown in Figure 4, this strain grows slowly in LB media at 28 C, and the cell growth is largely recovered by expression of the wide-type TopA. Similar to the wide-type TopA, expression of the TopA ZM3 variant in RFM475 also can restore the cell growth (Fig. 4). In contrast, expression of TopA ZM1 or ZM2 variant
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Figure 2. The iron-binding activity of the first two zinc-binding motifs in E. coli TopA. (A) Structural organization of E. coli TopA and constructed TopA fragments. (B) UV–Vis absorption spectra of E. coli TopA and TopA fragments purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. The concentration of the proteins was about 20 lM. (C) The iron content of E. coli TopA and TopA fragments purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. Results were the mean values plus or minus standard deviations from three independent experiments.
only partially restores the cell growth, and the ZM1/ ZM2 double mutant fails to restore the cell growth (Fig. 4). These results nicely correlate with the in vitro topoisomerase activity analyses of the TopA variants [Fig. 3(A)], and further suggest that the first two zinc-binding motifs in TopA have a crucial role for the topoisomerase activity.
Iron binding in the first two zinc-binding motifs of TopA inactivates its topoisomerase activity To further explore the effect of iron binding on the enzyme activity of TopA, we re-evaluated the competition of iron and zinc binding in the TopA ZM3 mutant which contains only the first two zincbinding motifs. In the experiments, TopA ZM3-mut protein was expressed in E. coli cells grown in the M9 minimal medium supplemented with a fixed concentration of zinc (0.15 lM) and increasing concentrations of iron (0–50 lM). As shown in Figure 5(A),
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when amounts of exogenous iron in M9 medium is gradually increased, the iron binding in TopA is progressively increased with concomitant decrease of zinc content in protein [Fig. 5(B)]. The topoisomerase activity of TopA ZM3-mut is also decreased accordingly [Fig. 5(C)]. Thus, iron can compete with zinc for the zinc-binding site in the first two zincbinding motifs and inhibit the topoisomerase activity of TopA.
Iron binding in TopA ZD domain decreases its ssDNA-binding activity The function of ZD domain in E. coli TopA was previously demonstrated. It has been shown to have strong ssDNA-binding activity and can interact with the passing single-strand DNA during relaxation of the negative supercoils.3 Therefore, it is reasonable to re-check whether the iron binding in ZD domain would have any effect on its ssDNA-binding affinity.
Iron Inhibits Escherichia coli Topoisomerase I Activity
Figure 3. The in vitro activity of E. coli TopA mutant proteins. (A) Topoisomerase activity of purified zinc bound E. coli TopA and TopA mutant proteins. Wide-type E. coli TopA and TopA mutant proteins were purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ZnSO4, and used for the topoisomerase activity assay. (B) Topoisomerase activity of iron bound purified E. coli TopA and TopA mutant proteins. Wide-type E. coli TopA and TopA mutant proteins were purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. Two protein concentrations (200 and 400 nM) were used for the enzyme activity analyses. The results were representatives from three independent experiments.
The iron-bound or zinc-bound wide-type ZD domain were expressed and purified from the E. coli cells grown in M9 media supplemented with exogenous iron or zinc, respectively. Meanwhile, ZM1/ZM2 double mutant ZD domain were also obtained similarly. As expected, the wide-type ZD domain, which was purified from E. coli cells grown in M9 media supplemented with exogenous iron, has red color and could bind iron (data not shown). However, the ZM1/ZM2-mut ZD domain hardly contains any iron under the same experimental conditions (data not shown). Then these proteins were assay for their ssDNA-binding activity. Figure 6(A) shows that the ssDNA-binding activity of iron-bound wide-type ZD domain is much weaker than that of the zinc-bound wide-type ZD domain. Interestingly, mutation of the first two zinc-binding motifs in ZD domain would also reduce its ssDNA-binding affinity. As shown in Figure 6(B), the ssDNA-binding activity of zincbound wide-type ZD domain is higher than that of the other three ones by 2–3-fold. These results suggest that iron binding in TopA may decrease the ssDNA-binding activity of ZD domain.
Iron binding in TopA changes the protein conformation Measurement of the intrinsic fluorescence emission of protein has been used to monitor protein confor-
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mational change.16,17 For E. coli TopA, fluorescence emission is altered when Mg (II) binds to catalytic site, Zn(II) is removed from the zinc-binding domain,18,19 or Cys662 (in the second Zn-binding motif) is mutated to either serine or histidine.20 Given that iron binding can inhibit the topoisomerase activity of TopA, it would be pertinent to explore the intrinsic tryptophan fluorescence change when TopA binds iron. The iron-bound or zinc-bound wide-type TopA were purified from the E. coli cells grown in M9 media supplemented with exogenous iron or zinc, respectively. Figure 7 shows that the intrinsic fluorescence of the iron-bound TopA is significantly less than that of the zinc-bound TopA. In parallel, the iron-treated (contains negligible amount of iron as presented in Fig. 1) or zinc-bound ZM1/ZM2 double mutant TopA proteins were also prepared from the E. coli cells and subjected to the intrinsic fluorescence measurements. As shown in Figure 7, both the iron-treated and zinc-bound ZM1/ZM2 mutant proteins have essentially the same intrinsic fluorescence as that of the iron-bound wide-type TopA, indicating that iron binding in TopA may result in a protein conformation that is similar to that of the inactive TopA ZM1/ZM2 double mutant protein. In addition, we also tested the intrinsic fluorescence of ZM1 and ZM2 single mutant under the same
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Figure 4. The in vivo activity of E. coli TopA mutant proteins. Complementation of wide-type E. coli TopA and TopA mutants in the topA-deleted E. coli strain RFM475. The overnight E. coli cell cultures were diluted to freshly prepared LB medium supplemented with ZnSO4 (50 lM) and ampicillin (100 lg/mL). Arabinose (0.005%, w/v) was added to induce the protein expression. Cells were grown at 28 C with vigorous shaking (300 rpm) for 8 h before cell density was rechecked. Results were the mean values plus or minus standard deviations from three independent experiments.
experimental conditions and found that the intrinsic fluorescence intensity of the iron-treated or zinc bound ZM1 and ZM2 mutants were significantly lower than the fully active wide-type zinc-bound TopA (results not shown), suggesting that mutations of ZM1, ZM2, or ZM1/ZM2 in TopA would result in conformational change of the proteins thus modulate enzyme activity.
Discussion In previous studies, we reported that E. coli TopA is able to bind both zinc and iron and that the ironbound TopA fails to relax the negatively supercoiled DNA in vitro.12 Here we find that TopA binds iron via the first two zinc-binding motifs in the ZD domain, and that the first two zinc-binding motifs have a crucial role for the topoisomerase activity in vitro and in vivo. Our studies further show that iron binding in the first two zinc-binding motifs may decrease the ssDNA-binding activity of ZD domain and change the protein structure, which would subsequently modulate the enzyme activity. The results substantiate the notion that iron binding in TopA may regulate the topoisomerase activity in E. coli cells. We were surprised to find that the iron binding in TopA is quite unique as compared to that of zinc binding. As shown in Figures 1 and 2, single mutation of either ZM1 or ZM2 would largely decrease
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the iron content in TopA, and the separated ZM1 or ZM2 peptide fragment could not bind iron unless they were together, indicating that both ZM1 and ZM2 are required for iron binding in TopA and that one atom of iron binds to TopA via the first two zincbinding motifs by occupying both of them simultaneously. Since there are totally eight cysteine residues clustered within the first two zinc-binding motifs, further investigations are needed to identify the specific coordinating residues for iron. Extensive studies have previously been carried out to determine the functional fragments in the C-terminal zinc-binding motifs in E. coli TopA. For example, it has been shown that truncation of the C-terminal fragment starting from amino acid residue 669 (including the second and third zinc-binding motifs) results in an inactive enzyme. Also, insertion of amino acids into the second zinc-binding motif dramatically attenuates enzyme activity,21 and single-point mutations of three cysteine residues (Cys619, Cys630, and Cys662) that belong to the first or second zinc-binding motif significantly decrease the enzyme activity of TopA.20 These results and the results from this study support the hypothesis that the first and the second zinc-binding motifs of the E. coli TopA are essential for the enzyme activity. While our results further suggest that the third zinc-binding motif is comparatively less crucial. However, the mechanism underlying the ironmediated inactivation of TopA is not immediately clear. It has been previously reported that the protein YrdD (a homologue of C-terminal E. coli TopA) is able to bind both iron and zinc, and only zincbound YrdD has strong ssDNA-binding affinity.22 It is very similar to the ZD domain in TopA as shown in Figure 6. Since the ssDNA-binding activity is quite important for the ZD domain in removal of negatively supercoiled DNA,3 we suggest that iron bound TopA is inactive partly because the iron binding may weaken the interaction of ZD domain with DNA substrate. Many zinc-binding proteins are also quite susceptible to toxic heavy metals, and the effects of toxic metals vary among different zinc-binding proteins.23–25 For example, the displacement of zinc by heavy metals may cause incomplete or incorrect coordination in zinc fingers, thus resulting in distortion of protein structure and inactivation of protein function.24 On the other hand, heavy metal binding in the zinc-binding sites may create a similar protein conformation and retain same enzyme activity. For example, the Cd(II)-bound TopA has essentially the same enzyme activity as the zinc-bound protein.26 Similarly, we found that the Co(II)-bound TopA is fully active to relax the negatively supercoiled DNA (unpublished data). In both cases, TopA binds three Cd(II) or Co(II) as Zn(II). The
Iron Inhibits Escherichia coli Topoisomerase I Activity
Figure 5. Iron and zinc competition in the first two zinc-binding motifs of E. coli TopA. (A) UV–Vis absorption spectra of TopA ZM3-mut proteins purified from E. coli cells grown in the M9 minimal medium supplemented with 0.15 lM ZnSO4 and 0, 1.0, 10.0, and 50 lM ferric citrate. The protein concentration was 40 lM. (B) Zinc content analyses of purified TopA ZM3-mut proteins. The zinc content in purified proteins was represented as the ratio of zinc to TopA ZM3-mut monomer. (C) The topoisomerase activity of TopA ZM3-mut proteins purified from E. coli cells grown in the M9 minimal medium supplemented with 0.15 lM ZnSO4 and 0, 1.0, 10.0, and 50 lM ferric citrate. Lane 1, no enzyme was added. The concentration of purified TopA ZM3-mut proteins used for the topoisomerase activity assay was 400 nM. The data are representatives from three independent experiments.
observation that the iron-bound TopA is inactive appears to be quite unique. Apparently, each TopA binds only one (not three) iron atom which can target both of the first two zinc-binding motifs, we therefore propose that iron binding in TopA can inhibit topoisomerase activity by disrupting the normal structural features of TopA protein via improper coordination with the first and second zinc-binding motifs.
Materials and Methods Plasmid construction and site-directed mutagenesis The plasmids pET-TopA, pET-TopA-N67, and pETTopA-ZD expressing the wide-type E. coli topoisomerase I (TopA), the N-terminal N67 domain (amino acids 1–597), and the C-terminal ZD domain (amino
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acids 598–865) were previously constructed.12 To express TopA without any tag, the DNA fragment encoding wide-type TopA was amplified from the E. coli genomic DNA with PCR using two primers: TopA-1 (50 -GAGGAATTAACCATGGGTAAAGCTCTT GTC-30 ) and TopA-2 (50 -CAAAACAGCCAAGCTT ATTTTTTTCCTTCAACCC-30 ). An In-Fusion HD Cloning Kit (Clontech) was used to subclone the PCR product seamlessly into an expression plasmid pBAD/HisA (Invitrogen) which was pre-digested by the restriction enzymes NcoI and HindIII. The site-directed mutagenesis was carried out using the Fast Mutagenesis System (TransGen). Three pairs of primers were designed to replace the first two cysteine residues with serine in each tetracysteine zinc finger in TopA, respectively, to generate three TopA variants [ZM1-mut (C599S/C602S), ZM2-mut (C662S/C665S), and ZM3-mut (C711S/C714S)]. A
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Figure 6. The ssDNA-binding activity of zinc bound and iron bound TopA-ZD. The 30 kDa ZD domain of TopA proteins were expressed and purified from E. coli cells grown in M9 medium with 50 lM ZnSO4 or with 50 lM ferric citrate. (A) The ssDNAbinding activity of Zn-bound wide-type TopA-ZD, Fe-bound wide-type TopA-ZD, Zn-bound ZM1/ZM2-mut TopA-ZD, Fe-bound ZM1/ZM2-mut TopA-ZD, and BSA (control). The proteins (1, 2, 4, 8 lM) were incubated with the fluorescein-labeled ssDNA (4 lM) at room temperature for 15 min. The samples were loaded onto a 0.8% agarose gel to resolve the protein-ssDNA complex from ‘‘free’’ ssDNA. (B) Comparison of the ssDNA-binding activity of Zn-bound wide-type TopA-ZD (rectangle), Fe-bound wide-type TopA-ZD (triangle), Zn-bound ZM1/ZM2-mut TopA-ZD (diamond), Fe-bound ZM1/ZM2-mut TopA-ZD (closed circle). The intensity of the ssDNA band was analyzed with ImageJ software and the amount of protein-bound ssDNA was compared to total ssDNA to obtain the ssDNA-binding percentage of each sample. The data are the averages with standard deviations from three independent experiments.
double mutant ZM1/ZM2-mut (C599S/C602S/C662S/ C665S) was obtained by an additional round of site-directed mutagenesis. Meanwhile, the ZD domain containing the same double mutation sites (ZD-ZM1/ZM2-mut) was also produced. For a truncated TopA protein in which the third zinc-binding motif and the followed 14 kDa C-terminal domain were removed (ZM3-del, amino acids 1–708), a pair of primers containing a stop codon at amino acid 709 was used for the site-directed mutagenesis. The plasmids expressing the first zinc-binding motif (ZM1, amino acids 598–659), the second zinc-binding motif (ZM2, amino acids 660–708), and the peptide containing both the first and the second zinc-binding
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motifs (ZM1 1 ZM2, amino acids 598–708) were also constructed into pET28b1 plasmids using the enzyme sites SacI and HindIII. The site-directed mutations and cloned DNA fragments were confirmed by direct sequencing.
Protein purification E. coli BL21 cells containing the pET-TopA, pETTopA-N67, pET-TopA-ZD, pET-ZM1-mut, pET-ZM2mut, pET-ZM3-mut, pET-ZM1/ZM2-mut, pET-ZDZM1/ZM2-mut, pET-ZM3-del, pET-ZM1, pET-ZM2, and pET-ZM11ZM2 were grown in either rich LB (Luria–Bertani) medium or the M9 minimal medium supplemented with glucose (0.2%), thiamin (5 lg/mL),
Iron Inhibits Escherichia coli Topoisomerase I Activity
were introduced into a topA-deleted E. coli cell strain RFM475 (a kind gift from Dr. Yuk-Ching TseDinh, Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY).14,15 The overnight cell cultures were diluted 1:100 to fresh LB medium supplemented with ZnSO4 (50 lM) and ampicillin (100 lg/mL). Arabinose (0.005%, w/v) was added to promote the expression of the wide-type TopA and TopA variants from the plasmids. The concentrations of cells were adjusted to O.D. of 0.02 at 600 nm and grew at 28 C with vigorous shaking (300 rpm) for 8 h before cell density was measured again. The relative growth rate was determined from the ratio of OD at 600 nm at 8 h to that at 0 h. Figure 7. Conformational change of E. coli TopA upon iron binding. TopA proteins were purified from E. coli cells grown in M9 medium with 50 lM ZnSO4 or with 50 lM ferric citrate. Intrinsic fluorescence spectra of zinc bound wide-type TopA (1) and ZM1/ZM2-mut TopA (2), as well as iron bound widetype TopA (3), and iron-treated ZM1/ZM2-mut TopA (4). Protein concentration was about 2 lM. The excitation wavelength was set at 280 nm.
and 20 amino acids (each at 10 lg/mL) to the O.D. of 1.0 at 600 nm before IPTG (isopropyl b-D-thiogalactoside) (0.2 mM) was added to induce protein expression at 37 C. The proteins were purified using the Ni–agarose column (Qiagen co.), followed by a gel filtration column (SuperdexTM 75 10/300GL, GE). The purity of purified protein was above 95% judging from the SDS/ PAGE gel stained by the Coomassie brilliant blue. The concentration of purified proteins was determined using Bradford Protein Assay Kit (Beyotime). The UV–Vis absorption spectra were measured in a Beckman DU800 UV–Vis spectrometer.
Topoisomerase activity assay The negatively supercoiled DNA relaxation assay was carried out following the procedure as described before.12 Briefly, purified wide-type TopA or TopA variants were incubated in reaction buffer (10 lL) containing Tris (50 mM, pH 8.0) and MgSO4 (3 mM) at 37 C for 10 min before the negatively supercoiled plasmid pUC18 DNA (250 ng) was added to reaction solutions. After incubation at 37 C for 30 min, reactions were stopped by adding 2.5 lL stop solution containing 5% SDS and 15% glycerol. The reaction solutions were then loaded on to a 1% agarose gel in TEA buffer. The agarose gel was run at 2 V/cm1 for 16 h, stained with ethidium bromide, and photographed.
Complementation of the wide-type TopA and TopA variants in the topA-deleted E. coli cell strain The plasmids pBAD-TopA, pBAD-ZM1-mut, pBADZM2-mut, pBAD-ZM3-mut, pBAD-ZM1/ZM2-mut
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Metal content analyses Total zinc content in protein samples was measured using the zinc indicator PAR (4-(2-pyridylazo)-resorcinol).27 The iron content in protein samples was determined using an iron indicator FerroZine.28 Zinc and iron contents in purified proteins were also analyzed by the inductively coupled plasma-emission spectrometry (ICP-ES). Both analyses produced similar results.
The DNA-binding activity assay The DNA-binding activity assay was carried out using a 35mer fluorescein labeled single-stranded oligonucleotide (50 -F*-GAAAACTCACAGGAAGCGGCCGAAGCGATTCGTCC-30 ). For the single-stranded DNA-binding assay, the fluorescein labeled 35mer was incubated with protein at room temperature for 15 min in a buffer containing Tris (20 mM, pH 8.0), NaCl (50 mM), b-mercaptoethanol (1 mM), MgSO4 (1 mM), and bovine serum albumin (BSA) (0.05 mg/mL). The samples were then loaded on to a 0.8% agarose gel in TEA (40 mM Tris acetate and 1 mM EDTA, pH 8.0) buffer. The agarose gel was run at 10 V per cm for 30 min and photographed in a Bio-Rad Gel DocTM XR1 Imaging System. The intensity of the DNA bands on agarose gel was quantified using the ImageJ software.
Intrinsic fluorescence measurement Purified proteins (2 lM) in buffer containing 20 mM Tris–HCl (pH 8.0) and 0.5M sodium chloride were preincubated at 37 C for 10 min. The intrinsic fluorescence of protein samples was measured in 3 mm path-length quartz microcuvettes using the Hitachi F-7000 fluorescence spectrophotometer. The emission spectra were monitored from 300 to 500 nm with the excitation at 280 nm. The spectrum of the buffer solution was used as a blank control.
Acknowledgments We thank Dr. Yuk-Ching Tse-Dinh (New York Medical College) for kindly providing the topA-deleted E. coli cell strain RFM475, and Dr. Huangen Ding
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(Louisiana State University) for the suggestions. This study was supported by the Chinese National Natural Science Foundation Grants (31228006, 31100576), and the Natural Science Foundation of Zhejiang Province Grants (LY13C050003, LY12C05003, LY13B070012).
15.
16.
References 1. Tse Y, Wang JC (1980) E. coli and M. luteus DNA topoisomerase I can catalyze catenation of decatenation of double-stranded DNA rings. Cell 22:269–276. 2. Wang JC (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3:430– 440. 3. Ahumada A, Tse-Dinh YC (2002) The role of the Zn(II) binding domain in the mechanism of E. coli DNA topoisomerase I. BMC Biochem 3:13. 4. Ahmed W, Bhat AG, Leelaram MN, Menon S, Nagaraja V (2013) Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage. Nucleic Acids Res 41:7462–7471. 5. Tse-Dinh YC, Beran-Steed RK (1988) Escherichia coli DNA topoisomerase I is a zinc metalloprotein with three repetitive zinc-binding domains. J Biol Chem 263:15857–15859. 6. Ahumada A, Tse-Dinh YC (1998) The Zn(II) binding motifs of E. coli DNA topoisomerase I is part of a highaffinity DNA binding domain. Biochem Biophys Res Commun 251:509–514. 7. Grishin NV (2000) C-terminal domains of Escherichia coli topoisomerase I belong to the zinc-ribbon superfamily. J Mol Biol 299:1165–1177. 8. Champoux JJ (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70:369– 413. 9. Cheng B, Zhu CX, Ji C, Ahumada A, Tse-Dinh YC (2003) Direct interaction between Escherichia coli RNA polymerase and the zinc ribbon domains of DNA topoisomerase I. J Biol Chem 278:30705–30710. 10. Lima CD, Wang JC, Mondragon A (1994) Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I. Nature 367:138–146. 11. Zhang Z, Cheng B, Tse-Dinh YC (2011) Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I. Proc Natl Acad Sci USA 108:6939–6944. 12. Lu J, Wang W, Tan G, Landry AP, Yi P, Si F, Ren Y, Ding H (2011) Escherichia coli topoisomerase I is an iron and zinc binding protein. Biometals 24:729–736. 13. Drolet M, Phoenix P, Menzel R, Masse E, Liu LF, Crouch RJ (1995) Overexpression of RNase H partially complements the growth defect of an Escherichia coli 䉭topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I. Proc Natl Acad Sci USA 92:3526–3530. 14. Qi H, Menzel R, Tse-Dinh YC (1999) Increased thermosensitivity associated with topoisomerase I deletion
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and promoter mutations in Escherichia coli. FEMS Microbiol Lett 178:141–146. Tse-Dinh YC (2000) Increased sensitivity to oxidative challenges associated with topA deletion in Escherichia coli. J Bacteriol 182:829–832. Hansen SB, Radic Z, Talley TT, Molles BE, Deerinck T, Tsigelny I, Taylor P (2002) Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein. J Biol Chem 277:41299–41302. James NG, Berger CL, Byrne SL, Smith VC, MacGillivray RT, Mason AB (2007) Intrinsic fluorescence reports a global conformational change in the N-lobe of human serum transferrin following iron release. Biochemistry 46:10603–10611. Samuel M, Zhu CX, Villanueva GB, Tse-Dinh YC (1993) Effect of zinc removal on the conformation of Escherichia coli DNA topoisomerase I. Arch Biochem Biophys 300:302–308. Zhu CX, Roche CJ, Tse-Dinh YC (1997) Effect of Mg(II) binding on the structure and activity of Escherichia coli DNA topoisomerase I. J Biol Chem 272:16206– 16210. Zhu CX, Qi HY, Tse-Dinh YC (1995) Mutation in Cys662 of Escherichia coli DNA topoisomerase I confers temperature sensitivity and change in DNA cleavage selectivity. J Mol Biol 250:609–616. Zumstein L, Wang JC (1986) Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis. J Mol Biol 191:333– 340. Cheng Z, Tan G, Wang W, Su X, Landry AP, Lu J, Ding H (2014) Iron and zinc binding activity of Escherichia coli topoisomerase I homolog YrdD. Biometals 27:229–236. Zawia NH, Crumpton T, Brydie M, Reddy GR, Razmiafshari M (2000) Disruption of the zinc finger domain: a common target that underlies many of the effects of lead. Neurotoxicology 21:1069–1080. Hartwig A (2001) Zinc finger proteins as potential targets for toxic metal ions: differential effects on structure and function. Antioxid Redox Signal 3:625–634. Hartwig A, Asmuss M, Blessing H, Hoffmann S, Jahnke G, Khandelwal S, Pelzer A, Burkle A (2002) Interference by toxic metal ions with zinc-dependent proteins involved in maintaining genomic stability. Food Chem Toxicol 40:1179–1184. Tse-Dinh YC (1991) Zinc (II) coordination in Escherichia coli DNA topoisomerase I is required for cleavable complex formation with DNA. J Biol Chem 266: 14317–14320. Bae JB, Park JH, Hahn MY, Kim MS, Roe JH (2004) Redox-dependent changes in RsrA, an anti-sigma factor in Streptomyces coelicolor: zinc release and disulfide bond formation. J Mol Biol 335:425–435. Cowart RE, Singleton FL, Hind JS (1993) A comparison of bathophenanthrolinedisulfonic acid and ferrozine as chelators of iron(II) in reduction reactions. Anal Biochem 211:151–155.
Iron Inhibits Escherichia coli Topoisomerase I Activity
Wu Wang,1 Xiaolu Su,1 Xiaobing Wang,2 Juanjuan Yang,1 Ting Zhang,1 Maofeng Wang,3 Rugen Wan,3 Guoqiang Tan,1 and Jianxin Lu1* 1
Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
2
Department of Rheumatism, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325035, China Department of Laboratory Medicine, Affiliated Dongyang Hospital of Wenzhou Medical University, Dongyang, Zhejiang 322100, China
3
Received 22 July 2014; Revised 26 August 2014; Accepted 27 August 2014 DOI: 10.1002/pro.2542 Published online 30 August 2014 proteinscience.org
Abstract: Escherichia coli DNA topoisomerase I (TopA) contains a 67 kDa N-terminal catalytic domain and a 30 kDa C-terminal zinc-binding region (ZD domain) which has three adjacent tetracysteine zinc-binding motifs. Previous studies have shown that E. coli TopA can bind both iron and zinc, and that iron binding in TopA results in failure to unwind the negatively supercoiled DNA. Here, we report that each E. coli TopA monomer binds one atom of iron via the first two zincbinding motifs in ZD domain and both the first and second zinc-binding motifs are required for iron binding in TopA. The site-directed mutagenesis studies further reveal that while the mutation of the third zinc-binding motif has very little effect on TopA’s activity, mutation of the first two zincbinding motifs in TopA greatly diminishes the topoisomerase activity in vitro and in vivo, indicating that the first two zinc-binding motifs in TopA are crucial for its function. The DNA-binding activity assay and intrinsic tryptophan fluorescence measurements show that iron binding in TopA may decrease the single-stranded (ss) DNA-binding activity of ZD domain and also change the protein structure of TopA, which subsequently modulate topoisomerase activity. Keywords: Escherichia coli topoisomerase I; iron binding; zinc-binding motif; topoisomerase activity
Introduction Abbreviations: TopA, E. coli topoisomerase I; PAR, 4-(2-pyridylazo)resorcinol; ZD, 30 kDa C-terminal zinc-binding domain in TopA. Grant sponsor: The Chinese National Natural Science Foundation Grants; Grant numbers: 31228006, 31100576; Grant sponsor: The Natural Science Foundation of Zhejiang Province Grants; Grant numbers: LY13C050003, LY12C05003, LY13B070012. *Correspondence to: Jianxin Lu; Key Laboratory of Laboratory Medicine, Ministry of Education of China, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China. E-mail: [email protected]
C 2014 The Protein Society Published by Wiley-Blackwell. V
Escherichia coli topoisomerase I (TopA) was the first discovered type I topoisomerase.1,2 Structurally, TopA contains a 67 kDa N-terminal catalytic domain (N67 domain) and a 30 kDa C-terminal zinc-binding region (ZD domain). The N-terminal domain binds double-stranded DNA and undertakes the cleavagerejoining catalytic action, but cannot complete relaxation of the negatively supercoiled DNA.3,4 The ZD domain comprises three tandem-arranged tetra-cysteine zinc-binding motifs and a 14 kDa DNA-binding domain,5–7 and is required not only for topoisomerase activity but also for interacting with RNA
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polymerase.8,9 Although crystal structures of the E. coli TopA N-terminal domain and the covalent intermediate complex interacting with single-stranded DNA have been reported,10,11 the full-length E. coli TopA crystal structure is currently not available. In previous studies, we found that E. coli TopA is able to bind both iron and zinc in E. coli cells, and that the iron-bound TopA fails to relax the negatively supercoiled DNA in vitro,12 suggesting that the enzyme activity of TopA may be regulated by intracellular iron content. Nevertheless, since the ZD domain contains three tandem-arranged tetracysteine zinc-binding motifs, the iron-binding sites in TopA remain elusive. Here we report that each E. coli TopA monomer binds one atom of iron via the first two tetracysteine zinc-binding motifs in the ZD domain. The site-directed mutagenesis studies further show that the first two zinc-binding motifs in TopA are essential for the topoisomerase activity in vitro and in vivo. Finally, iron binding in TopA appears to dramatically decrease the ssDNA-binding activity of ZD domain and the intrinsic fluorescence intensity of tryptophan in TopA protein, suggesting that iron binding in TopA may change the protein structure of TopA and subsequently modulate enzyme activity.
Results Each TopA monomer binds one atom of iron via the first two zinc-binding motifs in the ZD domain Previous studies indicated that E. coli TopA can bind both iron and zinc.12 However, the iron-binding sites in TopA are not known. Since TopA contains three zinc-binding motifs in the ZD domain,5 we constructed three TopA mutants (ZM1-mut, ZM2-mut, and ZM3-mut) in which the first two cysteine residues in the first (ZM1), second (ZM2), and third (ZM3) zinc-binding motif were replaced with serine, respectively. The wide-type TopA and each of TopA mutant proteins were expressed in the E. coli cells grown in the M9 minimal media supplemented with ferric citrate, and purified as described previously.12 The UV–Vis absorption analyses showed that the purified wide-type TopA has two major absorption peaks at 482 and 563 nm which indicate the iron binding in the protein.12 Purified ZM3-mut protein also has a similar absorption spectrum [Fig. 1(B)] and reddish color [Fig. 1(A)] as the wide-type TopA. In contrast, purified ZM1-mut and ZM2-mut have very little or no absorption peaks at 482 and 563 nm [Fig. 1(B)] and no color [Fig. 1(A)]. The total iron content analyses further revealed that wide-type TopA and ZM3-mut protein contain around one atom of iron per protein monomer, but the iron binding in ZM1 and ZM2 mutant proteins is significantly decreased [Fig. 1(C)]. We further constructed a TopA
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mutant in which both the first and second zincbinding motifs are mutated, and found that the iron content of the ZM1/ZM2 double mutant is essentially eliminated (Fig. 1). These results suggest that each wide-type TopA monomer can only bind one iron atom via the first and the second zinc-binding motifs in the ZD domain. In parallel, the wide-type TopA and each of TopA mutant proteins were expressed in the E. coli cells grown in the M9 minimal media supplemented with zinc sulfate. The measurement of zinc content shows that each wide-type TopA molecule contains three atoms of zinc as previously reported.5 In contrast, each of the three single mutants (ZM1-mut, ZM2-mut, and ZM3-mut) has two Zn(II) bound per monomer, and there is only one bound in ZM1/ZM2 double mutant [Fig. 1(D)]. The results indicate that the coordinations of Zn(II) with zinc-binding motifs in TopA is different from that of iron binding.
Both the first and the second zinc-binding motifs are required for iron binding in TopA To further explore the iron-binding sites in TopA, we subcloned the DNA fragments that encode a truncated TopA protein with deletion of the third zincbinding motif (ZM3-del), the N-terminal domain of TopA (N67), the first zinc-binding motif (ZM1), the second zinc-binding motif (ZM2), and the peptide fragment containing the first two zinc-binding motifs (ZM11ZM2) [Fig. 2(A)]. Each of these TopA fragments was successfully expressed in the E. coli cells grown in the M9 minimal media supplemented with ferric citrate. As shown in Figure 2B and C, purified ZM3-del has the same absorption peaks at 482 and 563 nm and contains almost the same amount of iron as the wild-type TopA. The ZM1 1 ZM2 fragment of TopA is also able to bind iron, although the iron content in the ZM1 1 ZM2 fragment is less than that in the intact TopA. In contrast, the purified Nterminal domain of TopA, the ZM1 and the ZM2 fragment contain negligible amount of iron [Fig. 2(B,C)]. Taken together, the results further suggest that E. coli TopA binds iron via the first and the second zinc-binding motifs, and both ZM1 and ZM2 are required for iron binding in E. coli TopA, while the third zinc-binding motif is not involve in the iron binding.
Disruption of the first two zinc-binding motifs in TopA results in an inactive enzyme in vitro and in vivo As only the first two zinc-binding motifs in the ZD domain are involved in the iron binding in TopA, it would be interesting to explore the specific roles of these zinc-binding motifs in the enzyme activity of TopA. To eliminate the effect of iron binding on topoisomerase activity of TopA,12 TopA and the TopA
Iron Inhibits Escherichia coli Topoisomerase I Activity
Figure 1. Iron-binding activity and zinc-binding activity of E. coli TopA and TopA mutants. (A) A photograph of E. coli TopA and TopA mutant proteins (40 lM) purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. (B) UV–Vis absorption spectra of purified E. coli TopA and TopA mutants. The concentration of the proteins was about 40 lM. (C) The iron content of E. coli TopA and TopA mutant proteins purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. (D) The zinc content of E. coli TopA and TopA mutant proteins purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ZnSO4. Results were the mean values plus or minus standard deviations from three independent experiments.
variants were prepared from the E. coli cells grown in the M9 minimal medium supplemented with 50 lM ZnSO4. Purified proteins were subjected to the in vitro topoisomerase activity assay as described previously.12 Figure 3(A) shows that the topoisomerase activity of ZM1-mut or ZM2-mut is dramatically decreased, while ZM3-mut is reduced by about 50%. When both the ZM1 and ZM2 are mutated, the TopA protein has no detectable activity [Fig. 3(A)], suggesting that the first two zinc-binding motifs are essential for the topoisomerase activity in vitro. TopA and the variants were also prepared from the E. coli cells grown in M9 minimal medium supplemented with 50 lM ferric citrate. As shown in Figure 3(B), the topoisomerase activity of the ironbound wide-type TopA, ZM1-mut, ZM2-mut, and ZM3-mut proteins are all inactive, consistent with the previous results that the iron-bound TopA is not active.12
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The enzyme activity of the TopA variants was further subjected to complementation analysis. The E. coli strain RFM475 lacks the topA gene but contains compensatory mutations in gyrB necessary for viability of RFM475, so it grows normally at 37 C. However, its survival would be threatened if the cells were exposed to high (52 C) and low (30 C) temperatures or oxidative stress.13–15 To test the in vivo function of the constructed TopA variants, the wild-type TopA and TopA variants were expressed in the RFM475 strain. The E. coli strain RFM475 expressing the wild-type TopA and TopA variants were grown in LB media at 28 C. As shown in Figure 4, this strain grows slowly in LB media at 28 C, and the cell growth is largely recovered by expression of the wide-type TopA. Similar to the wide-type TopA, expression of the TopA ZM3 variant in RFM475 also can restore the cell growth (Fig. 4). In contrast, expression of TopA ZM1 or ZM2 variant
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Figure 2. The iron-binding activity of the first two zinc-binding motifs in E. coli TopA. (A) Structural organization of E. coli TopA and constructed TopA fragments. (B) UV–Vis absorption spectra of E. coli TopA and TopA fragments purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. The concentration of the proteins was about 20 lM. (C) The iron content of E. coli TopA and TopA fragments purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. Results were the mean values plus or minus standard deviations from three independent experiments.
only partially restores the cell growth, and the ZM1/ ZM2 double mutant fails to restore the cell growth (Fig. 4). These results nicely correlate with the in vitro topoisomerase activity analyses of the TopA variants [Fig. 3(A)], and further suggest that the first two zinc-binding motifs in TopA have a crucial role for the topoisomerase activity.
Iron binding in the first two zinc-binding motifs of TopA inactivates its topoisomerase activity To further explore the effect of iron binding on the enzyme activity of TopA, we re-evaluated the competition of iron and zinc binding in the TopA ZM3 mutant which contains only the first two zincbinding motifs. In the experiments, TopA ZM3-mut protein was expressed in E. coli cells grown in the M9 minimal medium supplemented with a fixed concentration of zinc (0.15 lM) and increasing concentrations of iron (0–50 lM). As shown in Figure 5(A),
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when amounts of exogenous iron in M9 medium is gradually increased, the iron binding in TopA is progressively increased with concomitant decrease of zinc content in protein [Fig. 5(B)]. The topoisomerase activity of TopA ZM3-mut is also decreased accordingly [Fig. 5(C)]. Thus, iron can compete with zinc for the zinc-binding site in the first two zincbinding motifs and inhibit the topoisomerase activity of TopA.
Iron binding in TopA ZD domain decreases its ssDNA-binding activity The function of ZD domain in E. coli TopA was previously demonstrated. It has been shown to have strong ssDNA-binding activity and can interact with the passing single-strand DNA during relaxation of the negative supercoils.3 Therefore, it is reasonable to re-check whether the iron binding in ZD domain would have any effect on its ssDNA-binding affinity.
Iron Inhibits Escherichia coli Topoisomerase I Activity
Figure 3. The in vitro activity of E. coli TopA mutant proteins. (A) Topoisomerase activity of purified zinc bound E. coli TopA and TopA mutant proteins. Wide-type E. coli TopA and TopA mutant proteins were purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ZnSO4, and used for the topoisomerase activity assay. (B) Topoisomerase activity of iron bound purified E. coli TopA and TopA mutant proteins. Wide-type E. coli TopA and TopA mutant proteins were purified from E. coli cells grown in the M9 minimal medium supplemented with 50 lM ferric citrate. Two protein concentrations (200 and 400 nM) were used for the enzyme activity analyses. The results were representatives from three independent experiments.
The iron-bound or zinc-bound wide-type ZD domain were expressed and purified from the E. coli cells grown in M9 media supplemented with exogenous iron or zinc, respectively. Meanwhile, ZM1/ZM2 double mutant ZD domain were also obtained similarly. As expected, the wide-type ZD domain, which was purified from E. coli cells grown in M9 media supplemented with exogenous iron, has red color and could bind iron (data not shown). However, the ZM1/ZM2-mut ZD domain hardly contains any iron under the same experimental conditions (data not shown). Then these proteins were assay for their ssDNA-binding activity. Figure 6(A) shows that the ssDNA-binding activity of iron-bound wide-type ZD domain is much weaker than that of the zinc-bound wide-type ZD domain. Interestingly, mutation of the first two zinc-binding motifs in ZD domain would also reduce its ssDNA-binding affinity. As shown in Figure 6(B), the ssDNA-binding activity of zincbound wide-type ZD domain is higher than that of the other three ones by 2–3-fold. These results suggest that iron binding in TopA may decrease the ssDNA-binding activity of ZD domain.
Iron binding in TopA changes the protein conformation Measurement of the intrinsic fluorescence emission of protein has been used to monitor protein confor-
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mational change.16,17 For E. coli TopA, fluorescence emission is altered when Mg (II) binds to catalytic site, Zn(II) is removed from the zinc-binding domain,18,19 or Cys662 (in the second Zn-binding motif) is mutated to either serine or histidine.20 Given that iron binding can inhibit the topoisomerase activity of TopA, it would be pertinent to explore the intrinsic tryptophan fluorescence change when TopA binds iron. The iron-bound or zinc-bound wide-type TopA were purified from the E. coli cells grown in M9 media supplemented with exogenous iron or zinc, respectively. Figure 7 shows that the intrinsic fluorescence of the iron-bound TopA is significantly less than that of the zinc-bound TopA. In parallel, the iron-treated (contains negligible amount of iron as presented in Fig. 1) or zinc-bound ZM1/ZM2 double mutant TopA proteins were also prepared from the E. coli cells and subjected to the intrinsic fluorescence measurements. As shown in Figure 7, both the iron-treated and zinc-bound ZM1/ZM2 mutant proteins have essentially the same intrinsic fluorescence as that of the iron-bound wide-type TopA, indicating that iron binding in TopA may result in a protein conformation that is similar to that of the inactive TopA ZM1/ZM2 double mutant protein. In addition, we also tested the intrinsic fluorescence of ZM1 and ZM2 single mutant under the same
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Figure 4. The in vivo activity of E. coli TopA mutant proteins. Complementation of wide-type E. coli TopA and TopA mutants in the topA-deleted E. coli strain RFM475. The overnight E. coli cell cultures were diluted to freshly prepared LB medium supplemented with ZnSO4 (50 lM) and ampicillin (100 lg/mL). Arabinose (0.005%, w/v) was added to induce the protein expression. Cells were grown at 28 C with vigorous shaking (300 rpm) for 8 h before cell density was rechecked. Results were the mean values plus or minus standard deviations from three independent experiments.
experimental conditions and found that the intrinsic fluorescence intensity of the iron-treated or zinc bound ZM1 and ZM2 mutants were significantly lower than the fully active wide-type zinc-bound TopA (results not shown), suggesting that mutations of ZM1, ZM2, or ZM1/ZM2 in TopA would result in conformational change of the proteins thus modulate enzyme activity.
Discussion In previous studies, we reported that E. coli TopA is able to bind both zinc and iron and that the ironbound TopA fails to relax the negatively supercoiled DNA in vitro.12 Here we find that TopA binds iron via the first two zinc-binding motifs in the ZD domain, and that the first two zinc-binding motifs have a crucial role for the topoisomerase activity in vitro and in vivo. Our studies further show that iron binding in the first two zinc-binding motifs may decrease the ssDNA-binding activity of ZD domain and change the protein structure, which would subsequently modulate the enzyme activity. The results substantiate the notion that iron binding in TopA may regulate the topoisomerase activity in E. coli cells. We were surprised to find that the iron binding in TopA is quite unique as compared to that of zinc binding. As shown in Figures 1 and 2, single mutation of either ZM1 or ZM2 would largely decrease
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the iron content in TopA, and the separated ZM1 or ZM2 peptide fragment could not bind iron unless they were together, indicating that both ZM1 and ZM2 are required for iron binding in TopA and that one atom of iron binds to TopA via the first two zincbinding motifs by occupying both of them simultaneously. Since there are totally eight cysteine residues clustered within the first two zinc-binding motifs, further investigations are needed to identify the specific coordinating residues for iron. Extensive studies have previously been carried out to determine the functional fragments in the C-terminal zinc-binding motifs in E. coli TopA. For example, it has been shown that truncation of the C-terminal fragment starting from amino acid residue 669 (including the second and third zinc-binding motifs) results in an inactive enzyme. Also, insertion of amino acids into the second zinc-binding motif dramatically attenuates enzyme activity,21 and single-point mutations of three cysteine residues (Cys619, Cys630, and Cys662) that belong to the first or second zinc-binding motif significantly decrease the enzyme activity of TopA.20 These results and the results from this study support the hypothesis that the first and the second zinc-binding motifs of the E. coli TopA are essential for the enzyme activity. While our results further suggest that the third zinc-binding motif is comparatively less crucial. However, the mechanism underlying the ironmediated inactivation of TopA is not immediately clear. It has been previously reported that the protein YrdD (a homologue of C-terminal E. coli TopA) is able to bind both iron and zinc, and only zincbound YrdD has strong ssDNA-binding affinity.22 It is very similar to the ZD domain in TopA as shown in Figure 6. Since the ssDNA-binding activity is quite important for the ZD domain in removal of negatively supercoiled DNA,3 we suggest that iron bound TopA is inactive partly because the iron binding may weaken the interaction of ZD domain with DNA substrate. Many zinc-binding proteins are also quite susceptible to toxic heavy metals, and the effects of toxic metals vary among different zinc-binding proteins.23–25 For example, the displacement of zinc by heavy metals may cause incomplete or incorrect coordination in zinc fingers, thus resulting in distortion of protein structure and inactivation of protein function.24 On the other hand, heavy metal binding in the zinc-binding sites may create a similar protein conformation and retain same enzyme activity. For example, the Cd(II)-bound TopA has essentially the same enzyme activity as the zinc-bound protein.26 Similarly, we found that the Co(II)-bound TopA is fully active to relax the negatively supercoiled DNA (unpublished data). In both cases, TopA binds three Cd(II) or Co(II) as Zn(II). The
Iron Inhibits Escherichia coli Topoisomerase I Activity
Figure 5. Iron and zinc competition in the first two zinc-binding motifs of E. coli TopA. (A) UV–Vis absorption spectra of TopA ZM3-mut proteins purified from E. coli cells grown in the M9 minimal medium supplemented with 0.15 lM ZnSO4 and 0, 1.0, 10.0, and 50 lM ferric citrate. The protein concentration was 40 lM. (B) Zinc content analyses of purified TopA ZM3-mut proteins. The zinc content in purified proteins was represented as the ratio of zinc to TopA ZM3-mut monomer. (C) The topoisomerase activity of TopA ZM3-mut proteins purified from E. coli cells grown in the M9 minimal medium supplemented with 0.15 lM ZnSO4 and 0, 1.0, 10.0, and 50 lM ferric citrate. Lane 1, no enzyme was added. The concentration of purified TopA ZM3-mut proteins used for the topoisomerase activity assay was 400 nM. The data are representatives from three independent experiments.
observation that the iron-bound TopA is inactive appears to be quite unique. Apparently, each TopA binds only one (not three) iron atom which can target both of the first two zinc-binding motifs, we therefore propose that iron binding in TopA can inhibit topoisomerase activity by disrupting the normal structural features of TopA protein via improper coordination with the first and second zinc-binding motifs.
Materials and Methods Plasmid construction and site-directed mutagenesis The plasmids pET-TopA, pET-TopA-N67, and pETTopA-ZD expressing the wide-type E. coli topoisomerase I (TopA), the N-terminal N67 domain (amino acids 1–597), and the C-terminal ZD domain (amino
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acids 598–865) were previously constructed.12 To express TopA without any tag, the DNA fragment encoding wide-type TopA was amplified from the E. coli genomic DNA with PCR using two primers: TopA-1 (50 -GAGGAATTAACCATGGGTAAAGCTCTT GTC-30 ) and TopA-2 (50 -CAAAACAGCCAAGCTT ATTTTTTTCCTTCAACCC-30 ). An In-Fusion HD Cloning Kit (Clontech) was used to subclone the PCR product seamlessly into an expression plasmid pBAD/HisA (Invitrogen) which was pre-digested by the restriction enzymes NcoI and HindIII. The site-directed mutagenesis was carried out using the Fast Mutagenesis System (TransGen). Three pairs of primers were designed to replace the first two cysteine residues with serine in each tetracysteine zinc finger in TopA, respectively, to generate three TopA variants [ZM1-mut (C599S/C602S), ZM2-mut (C662S/C665S), and ZM3-mut (C711S/C714S)]. A
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Figure 6. The ssDNA-binding activity of zinc bound and iron bound TopA-ZD. The 30 kDa ZD domain of TopA proteins were expressed and purified from E. coli cells grown in M9 medium with 50 lM ZnSO4 or with 50 lM ferric citrate. (A) The ssDNAbinding activity of Zn-bound wide-type TopA-ZD, Fe-bound wide-type TopA-ZD, Zn-bound ZM1/ZM2-mut TopA-ZD, Fe-bound ZM1/ZM2-mut TopA-ZD, and BSA (control). The proteins (1, 2, 4, 8 lM) were incubated with the fluorescein-labeled ssDNA (4 lM) at room temperature for 15 min. The samples were loaded onto a 0.8% agarose gel to resolve the protein-ssDNA complex from ‘‘free’’ ssDNA. (B) Comparison of the ssDNA-binding activity of Zn-bound wide-type TopA-ZD (rectangle), Fe-bound wide-type TopA-ZD (triangle), Zn-bound ZM1/ZM2-mut TopA-ZD (diamond), Fe-bound ZM1/ZM2-mut TopA-ZD (closed circle). The intensity of the ssDNA band was analyzed with ImageJ software and the amount of protein-bound ssDNA was compared to total ssDNA to obtain the ssDNA-binding percentage of each sample. The data are the averages with standard deviations from three independent experiments.
double mutant ZM1/ZM2-mut (C599S/C602S/C662S/ C665S) was obtained by an additional round of site-directed mutagenesis. Meanwhile, the ZD domain containing the same double mutation sites (ZD-ZM1/ZM2-mut) was also produced. For a truncated TopA protein in which the third zinc-binding motif and the followed 14 kDa C-terminal domain were removed (ZM3-del, amino acids 1–708), a pair of primers containing a stop codon at amino acid 709 was used for the site-directed mutagenesis. The plasmids expressing the first zinc-binding motif (ZM1, amino acids 598–659), the second zinc-binding motif (ZM2, amino acids 660–708), and the peptide containing both the first and the second zinc-binding
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motifs (ZM1 1 ZM2, amino acids 598–708) were also constructed into pET28b1 plasmids using the enzyme sites SacI and HindIII. The site-directed mutations and cloned DNA fragments were confirmed by direct sequencing.
Protein purification E. coli BL21 cells containing the pET-TopA, pETTopA-N67, pET-TopA-ZD, pET-ZM1-mut, pET-ZM2mut, pET-ZM3-mut, pET-ZM1/ZM2-mut, pET-ZDZM1/ZM2-mut, pET-ZM3-del, pET-ZM1, pET-ZM2, and pET-ZM11ZM2 were grown in either rich LB (Luria–Bertani) medium or the M9 minimal medium supplemented with glucose (0.2%), thiamin (5 lg/mL),
Iron Inhibits Escherichia coli Topoisomerase I Activity
were introduced into a topA-deleted E. coli cell strain RFM475 (a kind gift from Dr. Yuk-Ching TseDinh, Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY).14,15 The overnight cell cultures were diluted 1:100 to fresh LB medium supplemented with ZnSO4 (50 lM) and ampicillin (100 lg/mL). Arabinose (0.005%, w/v) was added to promote the expression of the wide-type TopA and TopA variants from the plasmids. The concentrations of cells were adjusted to O.D. of 0.02 at 600 nm and grew at 28 C with vigorous shaking (300 rpm) for 8 h before cell density was measured again. The relative growth rate was determined from the ratio of OD at 600 nm at 8 h to that at 0 h. Figure 7. Conformational change of E. coli TopA upon iron binding. TopA proteins were purified from E. coli cells grown in M9 medium with 50 lM ZnSO4 or with 50 lM ferric citrate. Intrinsic fluorescence spectra of zinc bound wide-type TopA (1) and ZM1/ZM2-mut TopA (2), as well as iron bound widetype TopA (3), and iron-treated ZM1/ZM2-mut TopA (4). Protein concentration was about 2 lM. The excitation wavelength was set at 280 nm.
and 20 amino acids (each at 10 lg/mL) to the O.D. of 1.0 at 600 nm before IPTG (isopropyl b-D-thiogalactoside) (0.2 mM) was added to induce protein expression at 37 C. The proteins were purified using the Ni–agarose column (Qiagen co.), followed by a gel filtration column (SuperdexTM 75 10/300GL, GE). The purity of purified protein was above 95% judging from the SDS/ PAGE gel stained by the Coomassie brilliant blue. The concentration of purified proteins was determined using Bradford Protein Assay Kit (Beyotime). The UV–Vis absorption spectra were measured in a Beckman DU800 UV–Vis spectrometer.
Topoisomerase activity assay The negatively supercoiled DNA relaxation assay was carried out following the procedure as described before.12 Briefly, purified wide-type TopA or TopA variants were incubated in reaction buffer (10 lL) containing Tris (50 mM, pH 8.0) and MgSO4 (3 mM) at 37 C for 10 min before the negatively supercoiled plasmid pUC18 DNA (250 ng) was added to reaction solutions. After incubation at 37 C for 30 min, reactions were stopped by adding 2.5 lL stop solution containing 5% SDS and 15% glycerol. The reaction solutions were then loaded on to a 1% agarose gel in TEA buffer. The agarose gel was run at 2 V/cm1 for 16 h, stained with ethidium bromide, and photographed.
Complementation of the wide-type TopA and TopA variants in the topA-deleted E. coli cell strain The plasmids pBAD-TopA, pBAD-ZM1-mut, pBADZM2-mut, pBAD-ZM3-mut, pBAD-ZM1/ZM2-mut
Wang et al.
Metal content analyses Total zinc content in protein samples was measured using the zinc indicator PAR (4-(2-pyridylazo)-resorcinol).27 The iron content in protein samples was determined using an iron indicator FerroZine.28 Zinc and iron contents in purified proteins were also analyzed by the inductively coupled plasma-emission spectrometry (ICP-ES). Both analyses produced similar results.
The DNA-binding activity assay The DNA-binding activity assay was carried out using a 35mer fluorescein labeled single-stranded oligonucleotide (50 -F*-GAAAACTCACAGGAAGCGGCCGAAGCGATTCGTCC-30 ). For the single-stranded DNA-binding assay, the fluorescein labeled 35mer was incubated with protein at room temperature for 15 min in a buffer containing Tris (20 mM, pH 8.0), NaCl (50 mM), b-mercaptoethanol (1 mM), MgSO4 (1 mM), and bovine serum albumin (BSA) (0.05 mg/mL). The samples were then loaded on to a 0.8% agarose gel in TEA (40 mM Tris acetate and 1 mM EDTA, pH 8.0) buffer. The agarose gel was run at 10 V per cm for 30 min and photographed in a Bio-Rad Gel DocTM XR1 Imaging System. The intensity of the DNA bands on agarose gel was quantified using the ImageJ software.
Intrinsic fluorescence measurement Purified proteins (2 lM) in buffer containing 20 mM Tris–HCl (pH 8.0) and 0.5M sodium chloride were preincubated at 37 C for 10 min. The intrinsic fluorescence of protein samples was measured in 3 mm path-length quartz microcuvettes using the Hitachi F-7000 fluorescence spectrophotometer. The emission spectra were monitored from 300 to 500 nm with the excitation at 280 nm. The spectrum of the buffer solution was used as a blank control.
Acknowledgments We thank Dr. Yuk-Ching Tse-Dinh (New York Medical College) for kindly providing the topA-deleted E. coli cell strain RFM475, and Dr. Huangen Ding
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(Louisiana State University) for the suggestions. This study was supported by the Chinese National Natural Science Foundation Grants (31228006, 31100576), and the Natural Science Foundation of Zhejiang Province Grants (LY13C050003, LY12C05003, LY13B070012).
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