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Structure and allosteric coupling of type Ⅱ antitoxin CopASO
Biochemical and Biophysical Research Communications 514 (2019) 1122e1127
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Structure and allosteric coupling of type Ⅱ antitoxin CopASO Rongjuan Zhao a, b, c, Quan Li a, b, c, Jingjing Zhang a, b, c, Fudong Li d, Jianyun Yao e, Jiahai Zhang d, Lin Liu a, b, c, Xiaoxue Wang e, **, Xuecheng Zhang a, b, c, * a
School of Life Sciences, Anhui University, Hefei, Anhui, 230601, China Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei, Anhui, 230601, China c Anhui Key Laboratory of Modern Biomanufacturing, Hefei, Anhui, 230601, China d School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230026, China e CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Institution of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou, Guangdong, 510301, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2019 Accepted 6 May 2019 Available online 14 May 2019
Toxin-antitoxin (TA) systems play critical roles in the environment adaptation of bacteria. Allosteric coupling between the N-terminal DNA-binding domain and the C-terminal toxin-binding domain of antitoxins contributes to conditional cooperativity in the functioning of type II TA. Herein, using circular dichroism (CD), nuclear magnetic resonance (NMR), X-ray crystallography, and size exclusion chromatography (SEC), the structure and DNA binding of CopASO, a newly identified type II antitoxin in Shewanella oneidensis, were investigated. Our data show that CopASO is a typical RHH antitoxin with an ordered N-terminal domain and a disordered C-terminal domain, and furthermore indicate that the Cterminal domain facilitates DNA binding of the N-terminal domain, which in turn induces the C-terminal domain to fold and associate. © 2019 Elsevier Inc. All rights reserved.
Keywords: Allosteric coupling Antitoxin CopASO Ribbon-helix-helix motif Shewanella oneidensis
1. Introduction Toxineantitoxin (TA) systems of bacteria are emerging as important players in environment adaptation. All TA systems contain a toxin that manipulates vital processes such as transcription, translation, DNA replication and membrane homeostasis, and an antitoxin that inhibits the effects of the toxin [1]. TA systems, including a large and diverse family of proteins and noncoding RNAs, can be broadly categorized into six types depending on the mechanism by which the antitoxin neutralizes the toxin and the nature of antitoxin [1,2]. In the type II system, which is most well-studied, less stable proteic antitoxin binds to the stable proteic toxin to inhibit toxin activity under normal conditions; while under
Abbreviations: CD, circular dichroism; CopASO-C, peptide containing the C-terminal residues 58e94 of CopASO; CopASO-N, recombinant peptide containing the Nterminal residues 1e58 of CopASO; NMR, nuclear magnetic resonance; RHH, ribbonhelix-helix; SEC, size exclusion chromatography; TA, toxin-antitoxin. * Corresponding author. School of Life Sciences, Anhui University, Hefei, Anhui, 230601, China. ** Corresponding author. E-mail addresses: [email protected] (X. Wang), [email protected] (X. Zhang). https://doi.org/10.1016/j.bbrc.2019.05.049 0006-291X/© 2019 Elsevier Inc. All rights reserved.
stress conditions, the antitoxin is rapidly proteolyticaly degraded, leaving free toxin to target RNA, DNA or DNA gyrase [1,3]. Most of type II toxins that have been examined act as endoribonucleases and interfere with the translation process, or as inhibitor of gyrase and affect DNA replication [4]. Besides directly interacting with the toxin, the antitoxin neutralizes the toxin effects through proteinDNA interaction and inhibiting the TA promoter activity, which controls the transcription of both antitoxin and toxin [4]. This inhibitory activity is enhanced or blocked when the antitoxin is in complex with the toxin [5e7]. In most cases, the antitoxin protein may be divided into two regions: the N-terminal DNA-binding domain and the C-terminal toxin-binding domain, which are interconnected by a flexible small loop or hinge-like region. The toxin-binding domain of antitoxin is usually natively unstructured and induced to fold upon binding to the toxin [8e11]. Toxin binding of the antitoxin may change the states of the DNA-binding domain and the antitoxin-DNA complex, leading to further transcription repression [12,13]. The structures of the DNA-binding domains can be classified into four main types, namely helix-turn-helix (HTH), ribbon-helix-helix (RHH), SpoVT/ AbrB-type, and Phd/YefM [4,14]. The RHH type, the most common DNA-binding motif of antitoxin, are arranged as two antiparallel bstrands which compose a ribbon, with each strand coming from
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one of two protein monomers. The b-strands are involved both in dimer formation and in specific interactions with the DNA [15,16]. The DNA-binding domains usually bind to inverted repeat of specific palindromic sequences [17,18]. Interestingly, allosteric coupling between the C-terminal toxin-binding domain and the Nterminal DNA-binding domain of some type Ⅱ antitoxins has been reported. For example, both the stability and DNA-binding activity of the N-terminal domain of Phd antitoxin of bacteriophage P1 were dramatically enhanced by binding of the toxin Doc to the disordered C-terminal domain, contributing to the conditional cooperativity mechanism [7,19]. RelB-RelE TA system showed similar conditional cooperativity as Phd-Doc [10], though RelB-RelE are unrelated to Phd-Doc in sequence and structure. Particularly, self-association of the C-terminal domain of RelB and its binding to RelE can enhance the DNA binding activity of the N-terminal domain [20]. However, more details of the allosteric coupling within RHH antitoxins especially effects of the N-terminal domain on the C-terminal domain are yet to be revealed. ParESO/CopASO is a newly discovered type II TA pair in the CP4So prophage in Shewanella oneidensis, a model microorganism of marine bacteria. In this TA system, the antitoxin CopASO neutralizs the toxicity of the toxin ParESO, through direct protein-protein interactions, which inhibits cell growth and results in filamentous growth and cell death. In addition, by binding to a DNA motif in the promoter region containing two inverted repeats [50 -GTANTAC(N)3GTANTAC-3’], CopASO represses transcription of ParESO/ CopASO operon and another TA system PemKSO/PemISO in megaplasmid pMR-1. Notably, CopASO homologs, as a component of a TA pair or as orphan antitoxins, are widely spread in Shewanella and other Proteobacteria [21]. In this study, we investigated the structures and DNA binding of the N- and C- terminal domains of CopASO separately and in the context of full-length protein using circular dichroism (CD), nuclear magnetic resonance (NMR), X-ray crystallography, and size exclusion chromatography (SEC). The results show that CopASO is a typical RHH antitoxin. Notably, the disordered C-terminal domain appears to facilitate the ordered N-terminal domain binding to the promoter DNA, while the DNA binding induces the C-terminal domain to fold and self-associate. These data provide an insight into the allosteric coupling in CopASO and other RHH antitoxins. 2. Materials and methods 2.1. Materials Fragment corresponds to the C-terminal domain of CopASO (residues 58e94, named CopASO-C) was purchased from ChinaPeptides (Shanghai, China). DNAs were purchased from General Biosystems Co. (Anhui, China) in single chain form, double chain DNA was prepared by denaturing the mixture of complementary single chains at 95 C for 5 min and then annealing it through natural cooling. Other reagents were of analytic purity. 2.2. Preparation of CopASO and CopASO-N CopASO was recombinantly prepared as described in a previous report [21]. Fragment corresponds to its N-terminal domain (residues 1e58, named CopASO-N) was prepared likewise. In brief, its gene fragment was subcloned from the CopASO clone into pET28b plasmid with a His 6 tag codes attached to the C terminus. The recombinant plasmid was transformed into Escherichia.coli BL21(DE3) and the transformers were screened. Positive colonies were selected and checked by sequencing. The host with the recombinant plasmid was incubated in Luria-Bertani media and induced to express the recombinant proteins by addition of IPTG.
Fig. 1. Structures of CopASO and its N- and C- terminal domains. (a) CD spectra of CopASO, CopASO-N and CopASO-C; (b) Superimposition of the 1He15N HSQC spectra of CopASO (red) and CopASO-N (blue); (c) Crystal structure of CopASO-N, with the two polypeptide chains of the dimer colored in red and green, respectively, and the N- and C- termini labeled. The image was made using Pymol (The PyMOL Molecular Graphics € dinger, LLC.). (For interpretation of the references to color in System, Version 1.8 Schro this figure legend, the reader is referred to the Web version of this article.)
The recombinantly expressed proteins were purified by affinity chromatography using Chelating Sepharose Fast Flow (GE healthcare, USA), and further purified using a Hiload 16/60 Superdex 75 column (GE healthcare, USA) for crystallization. Particularly, proteins for NMR experiments were prepared in the same way except for that the host was incubated in M9 media with 15N NH4Cl as the only nitrogen source.
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2.3. Circular dichroism The samples for CD measurements contained 0.15 mg mL1 proteins, and four times concentration of DNA (for detection of DNA binding), in 20 mM sodium phosphate, 500 mM NaCl, pH 6.7. The measurements were performed on a Jasco Model J-810 spectropolarimeter (Jasco, Japan) at room temperature (20 C). Spectra were recorded in the range of 190e250 nm using a 0.1 cm path-length cell. Each sample was scanned three times to obtain an average. 2.4. Nuclear magnetic resonance The samples of free proteins contained 0.5 mM proteins, 20 mM NaH2PO4, 100 mM NaCl, and 10% D2O, in 500 mL volume and at pH 6.7. In the titration experiments, 0.18 mM DNA in the same buffer was added into 400 mL 0.3 mM protein solution in 8 times, 20 mL each times except for 60 mL the last time, and 1He15N HSQC spectrum was record before and after every addition. All NMR data were collected on a Bruker DMX600 spectrometer at 20 C. 2.5. X-ray crystallography Initial screening was carried out by the sitting-drop vapourdiffusion using Index Ⅰ and Ⅱ and Crystal Screen I kits (Hampton
Research, USA) at 4 C, with 1 mL protein solution (5 mg mL1 in the buffer of 20 mM sodium phosphate, 500 mM NaCl, pH 6.7) mixed with 1 mL reservoir solution. After several days, diffraction quality single crystals were obtained for CopASO-N in the condition of Index™ 90 (0.2 M sodium formate, 20% w/v polyethylene glycol 3350). The crystals of CopASO-N were harvested and soaked in a cryoprotectant solution consisting of 0.2 M sodium formate, 20% w/v polyethyleneglycol 3350, and 25% v/v glycerol for several seconds. The crystals were flash-cooled in liquid nitrogen and used for X-ray diffraction data collection using synchrotron radiation at 100 K on beamline BL19U1 at the SSRF (Shanghai, China). A diffraction data set was collected from one crystal with an oscillation angle of 1 per image. Diffraction data were indexed and integrated using the program MOSFLM [22] and scaled by SCALA in the CCP4 Program Suite [23]. The structure was solved by molecular replacement using the program MOLREP [24] employing the structure of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein (PDB 2gpe) as the search model. The initial model was further build and refined by Coot [25] and Phenix [26]. The structure was analyzed using Pymol €dinger, (The PyMOL Molecular Graphics System, Version 1.8 Schro LLC.) and the coordinates were deposited in Protein Data Bank with accession number 6IYA. Data-collection and refinement statistics are listed in Table S1.
Fig. 2. Size exclusion chromatography profiles for (a) CopASO, (b) CopASO-N, (c) CopASO-C and (d, e, and f) their mixture with (g) the promoter DNA on Hiload 16/60 Superdex75. In the mixture, the molar concentration of DNA was 4 times of the protein.
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Table 1 MWs and postulated compositions of the samples of CopASO, CopASO-N, CopASO-C and the promoter DNA and their mixture, assessed by SEC on Hiload 16/60 Superdex75. Sample
Retention volume (mL)
Theoretical MW (MWt, kDa)
Estimated MW by SEC (MWa, kDa)
MWa/MWt
Postulated composition
CopASO CopASO-N CopASO-C DNA CopASO þ DNA CopASO-N þ DNA CopASO-C þ DNA
12.1 13.0 16.4 13.0 10.3
12.0 8.0 4.1
32.4 21.1 4.1
2.7 2.6 1.0
Dimer Dimer Monomer
12.0 4 þ11.4 8.0 2 þ11.4 4 /11.4
76.8
1.4
29.5
1.1
4 /11.4
1.0 /1.0
Tetrameric protein þ monomeric DNA Dimeric protein þ monomeric DNA Monomeric protein /monomeric DNA
12.3 16.4 /13
2.6. Size exclusion chromatography SEC assays were performed on a Hiload 16/60 Superdex75 column (GE Healthcare, USA), which was equilibrated with the same buffer as the samples and eluted with the same buffer at a flow rate of 1 mL min1. The buffer of the samples was the same as that used in CD experiments. The solutes were monitored by ultraviolet absorbance at 216, 260, and 280 nm. For assessment of the MWs of the solutes, a standard sample (Gel Filtration Standard) containing thyroglobulin, g-globulin, ovalbumin, myoglobin, vitamin B12 in the same buffer was assayed as a reference (Fig. S1). According to the retention volumes of ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1.35 kDa), a regulation of Y ¼ 0.208Xþ4.028, where Y stood for LogMW and X for retention volume, was obtained by fitting. 3. Results 3.1. The N-terminal domain of CopASO is ordered and the C-terminal domain is disordered The N- and C- terminal domains of CopASO, based on the tertiary structure modeled by SWISS-MODEL, were supposed to contain residues 1e57 and 61e94 respectively (data not shown). Thus fragments containing residues 1e58 and 58e94 of CopASO, referred to as CopASO-N and CopASO-C respectively, were adopted to represent isolated N- and C- terminal domains in this study. The secondary structures of CopASO, CopASO-N and CopASO-C were evaluated by CD. There were two troughs near 208 nm and 222 nm in the CD spectra of CopASO and CopASO-N and a single trough near 200 nm in the spectrum of CopASO-C (Fig. 1a), denoting full length CopASO and CopASO-N consist mainly of helical structure, while CopASO-C is unstructured. To explore the states of the N- and Cterminal domains in the context of full length CopASO, 1He15N HSQC NMR spectrum of CopASO was recorded. The resonances in the spectrum were much less than the residue number of the protein, with some peaks broadened and overlapping each other (Fig. 1b). This indicated that some regions of the protein were not well folded and exchanged between different conformations. For comparison, 1He15N HSQC spectrum of CopASO-N was also recorded (we had tried to recombinantly produce 15N labeled CopASO-C for NMR, but failed). As expected, the spectrum displayed relatively evenly dispersed resonances, whose number was close to that of the residues of CopASO-N. When the 1He15N HSQC spectrum of CopASO-N was superimposed on that of CopASO, most of the resonances in the former were almost overlapped with or nearby those in the latter (Fig. 1b). These NMR data indicated that most of the Nterminal domain in the full length protein, like in the isolated form, was well folded, while the C-terminal domain in the full length protein was unstructured and flexible, consistent with the CD data. Reasonably due to the disordered and flexible properties of the C-terminal domain of CopASO, our attempt to crystallize the full
length protein failed. However, we succeeded in crystallizing ordered CopASO-N. The structure of CopASO-N was then solved by Xray diffracting of the crystal (Table S1). The crystal structure showed that free CopASO-N was a dimer and adopted a typical RHH fold, with residues 4e11 forming a b-strand and 13e25 and 30e47 forming two a-helices (Fig. 1c). As in other RHH antitoxins, the Nterminal domain of CopASO was dimerized by the intermolecular hydrogen bonding between the b-strands, which formed a ribbon. Notably, residues 50e58 were invisible in the electron density map. Using DALI [27], the structure of CopASO-N was compared with other RHH containing proteins. CopASO-N shares the highest structural similarity with the DNA-binding domain of Escherichia coli proline utilization A flavoprotein (PDB 2gpe-D) [28], with a sequence identity of 30% and a structural RMSD of 1.6 Å. Among structurally characterized antitoxins, MazE (PDB 5xe3-E) [29] shares the highest similarity with CopASO-N, with a sequence identity of 19% and a RMSD of 1.8 Å. 3.2. The C-terminal domain of CopASO facilitates the DNA binding of the N-terminal domain Based on the inverted repeat sequence 50 -GTANTACN3GTANTAC-30 in the promoter of ParESO/CopASO, we designed a DNA with complementary sequences of 50 -GGTATTACCTAGTAGTACT-30 and 50 -AGTACTACTAGGTAATACC-30 for DNA binding activity measurement of CopASO. CopASO, CopASO-N, CopASO-C and their mixture with the promoter DNA were first assayed by SEC. The elution volumes of the solutes were compared with those of standard proteins (Fig. S1) to assess their apparent MWs. As shown in Fig. 2
Fig. 3. 1He15N HSQC spectra of CopASO-N before (red) and after (blue and green) addition of the promoter DNA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 4. CD spectra of (a) CopASO, (b) CopASO-N, and (c) CopASO-C in the absence and presence of 4 times promoter DNA.
and Table 1, CopASO and CopASO-N were eluted with apparent MWs of 32.4 kDa and 21.1 kDa, 2.8 and 2.6 times of the theoretical MWs 12.0 kDa and 8.0 kDa, respectively. This indicated that when in free, both the full length protein and the N-terminal domain were dimerized, like other type Ⅱ and RHH antitoxins [7,19,20]. The main component of the mixture of CopASO and the DNA was eluted with an apparent MW of 76.8 kDa, close to a complex composed of four CopASO and one DNA, 59.4 kDa. The main component of the mixture of CopASO-N and the DNA was eluted with an apparent MW of 29.5 kDa, close to a complex of two CopASO-N and one DNA, 27.4 kDa. These indicated that binding of the CopASO dimer, via the N-terminal domains, to the DNA may facilitate its further dimerization, via the C-terminal domains, as in other RHH antitoxins such as RelB [10,20]. An alternative explanation is association between the C-terminal domains promotes the N-terminal domain binding to the DNA, as shown for Phd [30]. However, the apparent MW of CopASO-C assessed by SEC was the same as its theoretical MW 4.1 kDa, denoting a monomer in free form. Therefore, in the context of full length CopASO, more likely binding of the N-terminal domain to the DNA allosterically induced the C-terminal domain to selfassociate. In addition, CopASO-C and the DNA were eluted separately in the mixture, denoting there was no interaction between them. Interaction of CopASO with the promoter DNA was also investigated by NMR. When the DNA was titrated into CopASO, the solution turned to turbid immediately, and the resonance intensities were decreased dramatically to unobservable (data not shown), indicating that strong interaction led to protein association and precipitation. When the titration was performed on CopASO-N, the solution kept clear and no peak shift was observed though the resonance intensities decreased gradually (Fig. 3), indicating weaker interaction. These supported that the C-terminal domain may facilitate the N-terminal domain binding to the DNA and the binding may in turn promote self-association of the C-terminal domain, consistent with the SEC data.
3.3. DNA binding of the N-terminal domain of CopASO induces the C-terminal domain to fold To confirm the allosteric effect of the N-terminal domain on the C-terminal domain in CopASO, CD spectra of CopASO, CopASO-N and CopASO-C in the presence of the promoter DNA were recorded. Addition of the DNA changed significantly the CD spectrum of CopASO (Fig. 4a), while affected slightly the CD spectrum of CopASON (Fig. 4b) and hardly CopASO-C (Fig. 4c). On addition of the DNA, full length CopASO formed more helical structure, signified by deeper troughs around 208 nm and 222 nm. Since the secondary structures of CopASO-N in free and in the presence of the DNA were close to each other, the additional helical structure induced in the
full length protein by addition of the DNA might form in the Cterminal domain. This indicated that binding of the N-terminal domain to the DNA allosterically induced the unstructured C-terminal domain to fold, which may further promote its selfassociation. 4. Discussion Our data here showed that CopASO exhibited typical characteristics of RHH antitoxin as follows. CopASO is intrinsically disordered in the C-terminal domain, while the N-terminal domain adopts the typical RHH fold; The protein is dimeric in free while tetramerized on binding to the DNA, in which the C-terminal domain may facilitate the dimer binding to DNA and DNA binding may in turn promote the C-terminal domain's self-association. All these features contribute to the conditional cooperativity of type Ⅱ TAs [7,10,19,20]. A property critical for the conditional cooperativity is the allosteric effect of the C-terminal domain, in both free and toxin-bound forms, on the DNA binding of the N-terminal domain of the antitoxins [7,19,20]. Our data here offer another example for the allosteric effect of the C-terminal domain on the N-terminal domain of antitoxin. Moreover, we propose here that the DNA binding of the N-terminal domain of the antitoxin could in turn induce the C-terminal domain to fold. The induced fold of the Cterminal domains may enhance its self-association as well as its interaction with the toxin, given that toxin-binding may induce the C-terminal domains to fold as well. As mentioned above, CopASO, at high concentration in the sample for NMR, associated and precipitated when titrated with the promoter DNA, thus detailed structural changes of the C-terminal domain caused by binding of the Nterminal domain to DNA was unable to be explored by NMR. Try of solving the structure of CopASO-DNA complex by X-Ray crystallography is ongoing. Author contributions XZ and XW conceived and supervised the study; RZ, XZ designed experiments; RZ, QL, and JZ performed experiments; JY provided materials, RZ, FL, LL and XZ analyzed data; RZ and XZ wrote the manuscript; LL and XW made manuscript revisions. Acknowledgements This work was supported by the National Natural Science Foundation of China [grant number 31470775]. Transparency document Transparency document related to this article can be found
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online at https://doi.org/10.1016/j.bbrc.2019.05.049. Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.049.
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Structure and allosteric coupling of type Ⅱ antitoxin CopASO Rongjuan Zhao a, b, c, Quan Li a, b, c, Jingjing Zhang a, b, c, Fudong Li d, Jianyun Yao e, Jiahai Zhang d, Lin Liu a, b, c, Xiaoxue Wang e, **, Xuecheng Zhang a, b, c, * a
School of Life Sciences, Anhui University, Hefei, Anhui, 230601, China Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei, Anhui, 230601, China c Anhui Key Laboratory of Modern Biomanufacturing, Hefei, Anhui, 230601, China d School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230026, China e CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Institution of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou, Guangdong, 510301, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 April 2019 Accepted 6 May 2019 Available online 14 May 2019
Toxin-antitoxin (TA) systems play critical roles in the environment adaptation of bacteria. Allosteric coupling between the N-terminal DNA-binding domain and the C-terminal toxin-binding domain of antitoxins contributes to conditional cooperativity in the functioning of type II TA. Herein, using circular dichroism (CD), nuclear magnetic resonance (NMR), X-ray crystallography, and size exclusion chromatography (SEC), the structure and DNA binding of CopASO, a newly identified type II antitoxin in Shewanella oneidensis, were investigated. Our data show that CopASO is a typical RHH antitoxin with an ordered N-terminal domain and a disordered C-terminal domain, and furthermore indicate that the Cterminal domain facilitates DNA binding of the N-terminal domain, which in turn induces the C-terminal domain to fold and associate. © 2019 Elsevier Inc. All rights reserved.
Keywords: Allosteric coupling Antitoxin CopASO Ribbon-helix-helix motif Shewanella oneidensis
1. Introduction Toxineantitoxin (TA) systems of bacteria are emerging as important players in environment adaptation. All TA systems contain a toxin that manipulates vital processes such as transcription, translation, DNA replication and membrane homeostasis, and an antitoxin that inhibits the effects of the toxin [1]. TA systems, including a large and diverse family of proteins and noncoding RNAs, can be broadly categorized into six types depending on the mechanism by which the antitoxin neutralizes the toxin and the nature of antitoxin [1,2]. In the type II system, which is most well-studied, less stable proteic antitoxin binds to the stable proteic toxin to inhibit toxin activity under normal conditions; while under
Abbreviations: CD, circular dichroism; CopASO-C, peptide containing the C-terminal residues 58e94 of CopASO; CopASO-N, recombinant peptide containing the Nterminal residues 1e58 of CopASO; NMR, nuclear magnetic resonance; RHH, ribbonhelix-helix; SEC, size exclusion chromatography; TA, toxin-antitoxin. * Corresponding author. School of Life Sciences, Anhui University, Hefei, Anhui, 230601, China. ** Corresponding author. E-mail addresses: [email protected] (X. Wang), [email protected] (X. Zhang). https://doi.org/10.1016/j.bbrc.2019.05.049 0006-291X/© 2019 Elsevier Inc. All rights reserved.
stress conditions, the antitoxin is rapidly proteolyticaly degraded, leaving free toxin to target RNA, DNA or DNA gyrase [1,3]. Most of type II toxins that have been examined act as endoribonucleases and interfere with the translation process, or as inhibitor of gyrase and affect DNA replication [4]. Besides directly interacting with the toxin, the antitoxin neutralizes the toxin effects through proteinDNA interaction and inhibiting the TA promoter activity, which controls the transcription of both antitoxin and toxin [4]. This inhibitory activity is enhanced or blocked when the antitoxin is in complex with the toxin [5e7]. In most cases, the antitoxin protein may be divided into two regions: the N-terminal DNA-binding domain and the C-terminal toxin-binding domain, which are interconnected by a flexible small loop or hinge-like region. The toxin-binding domain of antitoxin is usually natively unstructured and induced to fold upon binding to the toxin [8e11]. Toxin binding of the antitoxin may change the states of the DNA-binding domain and the antitoxin-DNA complex, leading to further transcription repression [12,13]. The structures of the DNA-binding domains can be classified into four main types, namely helix-turn-helix (HTH), ribbon-helix-helix (RHH), SpoVT/ AbrB-type, and Phd/YefM [4,14]. The RHH type, the most common DNA-binding motif of antitoxin, are arranged as two antiparallel bstrands which compose a ribbon, with each strand coming from
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one of two protein monomers. The b-strands are involved both in dimer formation and in specific interactions with the DNA [15,16]. The DNA-binding domains usually bind to inverted repeat of specific palindromic sequences [17,18]. Interestingly, allosteric coupling between the C-terminal toxin-binding domain and the Nterminal DNA-binding domain of some type Ⅱ antitoxins has been reported. For example, both the stability and DNA-binding activity of the N-terminal domain of Phd antitoxin of bacteriophage P1 were dramatically enhanced by binding of the toxin Doc to the disordered C-terminal domain, contributing to the conditional cooperativity mechanism [7,19]. RelB-RelE TA system showed similar conditional cooperativity as Phd-Doc [10], though RelB-RelE are unrelated to Phd-Doc in sequence and structure. Particularly, self-association of the C-terminal domain of RelB and its binding to RelE can enhance the DNA binding activity of the N-terminal domain [20]. However, more details of the allosteric coupling within RHH antitoxins especially effects of the N-terminal domain on the C-terminal domain are yet to be revealed. ParESO/CopASO is a newly discovered type II TA pair in the CP4So prophage in Shewanella oneidensis, a model microorganism of marine bacteria. In this TA system, the antitoxin CopASO neutralizs the toxicity of the toxin ParESO, through direct protein-protein interactions, which inhibits cell growth and results in filamentous growth and cell death. In addition, by binding to a DNA motif in the promoter region containing two inverted repeats [50 -GTANTAC(N)3GTANTAC-3’], CopASO represses transcription of ParESO/ CopASO operon and another TA system PemKSO/PemISO in megaplasmid pMR-1. Notably, CopASO homologs, as a component of a TA pair or as orphan antitoxins, are widely spread in Shewanella and other Proteobacteria [21]. In this study, we investigated the structures and DNA binding of the N- and C- terminal domains of CopASO separately and in the context of full-length protein using circular dichroism (CD), nuclear magnetic resonance (NMR), X-ray crystallography, and size exclusion chromatography (SEC). The results show that CopASO is a typical RHH antitoxin. Notably, the disordered C-terminal domain appears to facilitate the ordered N-terminal domain binding to the promoter DNA, while the DNA binding induces the C-terminal domain to fold and self-associate. These data provide an insight into the allosteric coupling in CopASO and other RHH antitoxins. 2. Materials and methods 2.1. Materials Fragment corresponds to the C-terminal domain of CopASO (residues 58e94, named CopASO-C) was purchased from ChinaPeptides (Shanghai, China). DNAs were purchased from General Biosystems Co. (Anhui, China) in single chain form, double chain DNA was prepared by denaturing the mixture of complementary single chains at 95 C for 5 min and then annealing it through natural cooling. Other reagents were of analytic purity. 2.2. Preparation of CopASO and CopASO-N CopASO was recombinantly prepared as described in a previous report [21]. Fragment corresponds to its N-terminal domain (residues 1e58, named CopASO-N) was prepared likewise. In brief, its gene fragment was subcloned from the CopASO clone into pET28b plasmid with a His 6 tag codes attached to the C terminus. The recombinant plasmid was transformed into Escherichia.coli BL21(DE3) and the transformers were screened. Positive colonies were selected and checked by sequencing. The host with the recombinant plasmid was incubated in Luria-Bertani media and induced to express the recombinant proteins by addition of IPTG.
Fig. 1. Structures of CopASO and its N- and C- terminal domains. (a) CD spectra of CopASO, CopASO-N and CopASO-C; (b) Superimposition of the 1He15N HSQC spectra of CopASO (red) and CopASO-N (blue); (c) Crystal structure of CopASO-N, with the two polypeptide chains of the dimer colored in red and green, respectively, and the N- and C- termini labeled. The image was made using Pymol (The PyMOL Molecular Graphics € dinger, LLC.). (For interpretation of the references to color in System, Version 1.8 Schro this figure legend, the reader is referred to the Web version of this article.)
The recombinantly expressed proteins were purified by affinity chromatography using Chelating Sepharose Fast Flow (GE healthcare, USA), and further purified using a Hiload 16/60 Superdex 75 column (GE healthcare, USA) for crystallization. Particularly, proteins for NMR experiments were prepared in the same way except for that the host was incubated in M9 media with 15N NH4Cl as the only nitrogen source.
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2.3. Circular dichroism The samples for CD measurements contained 0.15 mg mL1 proteins, and four times concentration of DNA (for detection of DNA binding), in 20 mM sodium phosphate, 500 mM NaCl, pH 6.7. The measurements were performed on a Jasco Model J-810 spectropolarimeter (Jasco, Japan) at room temperature (20 C). Spectra were recorded in the range of 190e250 nm using a 0.1 cm path-length cell. Each sample was scanned three times to obtain an average. 2.4. Nuclear magnetic resonance The samples of free proteins contained 0.5 mM proteins, 20 mM NaH2PO4, 100 mM NaCl, and 10% D2O, in 500 mL volume and at pH 6.7. In the titration experiments, 0.18 mM DNA in the same buffer was added into 400 mL 0.3 mM protein solution in 8 times, 20 mL each times except for 60 mL the last time, and 1He15N HSQC spectrum was record before and after every addition. All NMR data were collected on a Bruker DMX600 spectrometer at 20 C. 2.5. X-ray crystallography Initial screening was carried out by the sitting-drop vapourdiffusion using Index Ⅰ and Ⅱ and Crystal Screen I kits (Hampton
Research, USA) at 4 C, with 1 mL protein solution (5 mg mL1 in the buffer of 20 mM sodium phosphate, 500 mM NaCl, pH 6.7) mixed with 1 mL reservoir solution. After several days, diffraction quality single crystals were obtained for CopASO-N in the condition of Index™ 90 (0.2 M sodium formate, 20% w/v polyethylene glycol 3350). The crystals of CopASO-N were harvested and soaked in a cryoprotectant solution consisting of 0.2 M sodium formate, 20% w/v polyethyleneglycol 3350, and 25% v/v glycerol for several seconds. The crystals were flash-cooled in liquid nitrogen and used for X-ray diffraction data collection using synchrotron radiation at 100 K on beamline BL19U1 at the SSRF (Shanghai, China). A diffraction data set was collected from one crystal with an oscillation angle of 1 per image. Diffraction data were indexed and integrated using the program MOSFLM [22] and scaled by SCALA in the CCP4 Program Suite [23]. The structure was solved by molecular replacement using the program MOLREP [24] employing the structure of the DNA-binding domain of Escherichia coli proline utilization A flavoprotein (PDB 2gpe) as the search model. The initial model was further build and refined by Coot [25] and Phenix [26]. The structure was analyzed using Pymol €dinger, (The PyMOL Molecular Graphics System, Version 1.8 Schro LLC.) and the coordinates were deposited in Protein Data Bank with accession number 6IYA. Data-collection and refinement statistics are listed in Table S1.
Fig. 2. Size exclusion chromatography profiles for (a) CopASO, (b) CopASO-N, (c) CopASO-C and (d, e, and f) their mixture with (g) the promoter DNA on Hiload 16/60 Superdex75. In the mixture, the molar concentration of DNA was 4 times of the protein.
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Table 1 MWs and postulated compositions of the samples of CopASO, CopASO-N, CopASO-C and the promoter DNA and their mixture, assessed by SEC on Hiload 16/60 Superdex75. Sample
Retention volume (mL)
Theoretical MW (MWt, kDa)
Estimated MW by SEC (MWa, kDa)
MWa/MWt
Postulated composition
CopASO CopASO-N CopASO-C DNA CopASO þ DNA CopASO-N þ DNA CopASO-C þ DNA
12.1 13.0 16.4 13.0 10.3
12.0 8.0 4.1
32.4 21.1 4.1
2.7 2.6 1.0
Dimer Dimer Monomer
12.0 4 þ11.4 8.0 2 þ11.4 4 /11.4
76.8
1.4
29.5
1.1
4 /11.4
1.0 /1.0
Tetrameric protein þ monomeric DNA Dimeric protein þ monomeric DNA Monomeric protein /monomeric DNA
12.3 16.4 /13
2.6. Size exclusion chromatography SEC assays were performed on a Hiload 16/60 Superdex75 column (GE Healthcare, USA), which was equilibrated with the same buffer as the samples and eluted with the same buffer at a flow rate of 1 mL min1. The buffer of the samples was the same as that used in CD experiments. The solutes were monitored by ultraviolet absorbance at 216, 260, and 280 nm. For assessment of the MWs of the solutes, a standard sample (Gel Filtration Standard) containing thyroglobulin, g-globulin, ovalbumin, myoglobin, vitamin B12 in the same buffer was assayed as a reference (Fig. S1). According to the retention volumes of ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1.35 kDa), a regulation of Y ¼ 0.208Xþ4.028, where Y stood for LogMW and X for retention volume, was obtained by fitting. 3. Results 3.1. The N-terminal domain of CopASO is ordered and the C-terminal domain is disordered The N- and C- terminal domains of CopASO, based on the tertiary structure modeled by SWISS-MODEL, were supposed to contain residues 1e57 and 61e94 respectively (data not shown). Thus fragments containing residues 1e58 and 58e94 of CopASO, referred to as CopASO-N and CopASO-C respectively, were adopted to represent isolated N- and C- terminal domains in this study. The secondary structures of CopASO, CopASO-N and CopASO-C were evaluated by CD. There were two troughs near 208 nm and 222 nm in the CD spectra of CopASO and CopASO-N and a single trough near 200 nm in the spectrum of CopASO-C (Fig. 1a), denoting full length CopASO and CopASO-N consist mainly of helical structure, while CopASO-C is unstructured. To explore the states of the N- and Cterminal domains in the context of full length CopASO, 1He15N HSQC NMR spectrum of CopASO was recorded. The resonances in the spectrum were much less than the residue number of the protein, with some peaks broadened and overlapping each other (Fig. 1b). This indicated that some regions of the protein were not well folded and exchanged between different conformations. For comparison, 1He15N HSQC spectrum of CopASO-N was also recorded (we had tried to recombinantly produce 15N labeled CopASO-C for NMR, but failed). As expected, the spectrum displayed relatively evenly dispersed resonances, whose number was close to that of the residues of CopASO-N. When the 1He15N HSQC spectrum of CopASO-N was superimposed on that of CopASO, most of the resonances in the former were almost overlapped with or nearby those in the latter (Fig. 1b). These NMR data indicated that most of the Nterminal domain in the full length protein, like in the isolated form, was well folded, while the C-terminal domain in the full length protein was unstructured and flexible, consistent with the CD data. Reasonably due to the disordered and flexible properties of the C-terminal domain of CopASO, our attempt to crystallize the full
length protein failed. However, we succeeded in crystallizing ordered CopASO-N. The structure of CopASO-N was then solved by Xray diffracting of the crystal (Table S1). The crystal structure showed that free CopASO-N was a dimer and adopted a typical RHH fold, with residues 4e11 forming a b-strand and 13e25 and 30e47 forming two a-helices (Fig. 1c). As in other RHH antitoxins, the Nterminal domain of CopASO was dimerized by the intermolecular hydrogen bonding between the b-strands, which formed a ribbon. Notably, residues 50e58 were invisible in the electron density map. Using DALI [27], the structure of CopASO-N was compared with other RHH containing proteins. CopASO-N shares the highest structural similarity with the DNA-binding domain of Escherichia coli proline utilization A flavoprotein (PDB 2gpe-D) [28], with a sequence identity of 30% and a structural RMSD of 1.6 Å. Among structurally characterized antitoxins, MazE (PDB 5xe3-E) [29] shares the highest similarity with CopASO-N, with a sequence identity of 19% and a RMSD of 1.8 Å. 3.2. The C-terminal domain of CopASO facilitates the DNA binding of the N-terminal domain Based on the inverted repeat sequence 50 -GTANTACN3GTANTAC-30 in the promoter of ParESO/CopASO, we designed a DNA with complementary sequences of 50 -GGTATTACCTAGTAGTACT-30 and 50 -AGTACTACTAGGTAATACC-30 for DNA binding activity measurement of CopASO. CopASO, CopASO-N, CopASO-C and their mixture with the promoter DNA were first assayed by SEC. The elution volumes of the solutes were compared with those of standard proteins (Fig. S1) to assess their apparent MWs. As shown in Fig. 2
Fig. 3. 1He15N HSQC spectra of CopASO-N before (red) and after (blue and green) addition of the promoter DNA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 4. CD spectra of (a) CopASO, (b) CopASO-N, and (c) CopASO-C in the absence and presence of 4 times promoter DNA.
and Table 1, CopASO and CopASO-N were eluted with apparent MWs of 32.4 kDa and 21.1 kDa, 2.8 and 2.6 times of the theoretical MWs 12.0 kDa and 8.0 kDa, respectively. This indicated that when in free, both the full length protein and the N-terminal domain were dimerized, like other type Ⅱ and RHH antitoxins [7,19,20]. The main component of the mixture of CopASO and the DNA was eluted with an apparent MW of 76.8 kDa, close to a complex composed of four CopASO and one DNA, 59.4 kDa. The main component of the mixture of CopASO-N and the DNA was eluted with an apparent MW of 29.5 kDa, close to a complex of two CopASO-N and one DNA, 27.4 kDa. These indicated that binding of the CopASO dimer, via the N-terminal domains, to the DNA may facilitate its further dimerization, via the C-terminal domains, as in other RHH antitoxins such as RelB [10,20]. An alternative explanation is association between the C-terminal domains promotes the N-terminal domain binding to the DNA, as shown for Phd [30]. However, the apparent MW of CopASO-C assessed by SEC was the same as its theoretical MW 4.1 kDa, denoting a monomer in free form. Therefore, in the context of full length CopASO, more likely binding of the N-terminal domain to the DNA allosterically induced the C-terminal domain to selfassociate. In addition, CopASO-C and the DNA were eluted separately in the mixture, denoting there was no interaction between them. Interaction of CopASO with the promoter DNA was also investigated by NMR. When the DNA was titrated into CopASO, the solution turned to turbid immediately, and the resonance intensities were decreased dramatically to unobservable (data not shown), indicating that strong interaction led to protein association and precipitation. When the titration was performed on CopASO-N, the solution kept clear and no peak shift was observed though the resonance intensities decreased gradually (Fig. 3), indicating weaker interaction. These supported that the C-terminal domain may facilitate the N-terminal domain binding to the DNA and the binding may in turn promote self-association of the C-terminal domain, consistent with the SEC data.
3.3. DNA binding of the N-terminal domain of CopASO induces the C-terminal domain to fold To confirm the allosteric effect of the N-terminal domain on the C-terminal domain in CopASO, CD spectra of CopASO, CopASO-N and CopASO-C in the presence of the promoter DNA were recorded. Addition of the DNA changed significantly the CD spectrum of CopASO (Fig. 4a), while affected slightly the CD spectrum of CopASON (Fig. 4b) and hardly CopASO-C (Fig. 4c). On addition of the DNA, full length CopASO formed more helical structure, signified by deeper troughs around 208 nm and 222 nm. Since the secondary structures of CopASO-N in free and in the presence of the DNA were close to each other, the additional helical structure induced in the
full length protein by addition of the DNA might form in the Cterminal domain. This indicated that binding of the N-terminal domain to the DNA allosterically induced the unstructured C-terminal domain to fold, which may further promote its selfassociation. 4. Discussion Our data here showed that CopASO exhibited typical characteristics of RHH antitoxin as follows. CopASO is intrinsically disordered in the C-terminal domain, while the N-terminal domain adopts the typical RHH fold; The protein is dimeric in free while tetramerized on binding to the DNA, in which the C-terminal domain may facilitate the dimer binding to DNA and DNA binding may in turn promote the C-terminal domain's self-association. All these features contribute to the conditional cooperativity of type Ⅱ TAs [7,10,19,20]. A property critical for the conditional cooperativity is the allosteric effect of the C-terminal domain, in both free and toxin-bound forms, on the DNA binding of the N-terminal domain of the antitoxins [7,19,20]. Our data here offer another example for the allosteric effect of the C-terminal domain on the N-terminal domain of antitoxin. Moreover, we propose here that the DNA binding of the N-terminal domain of the antitoxin could in turn induce the C-terminal domain to fold. The induced fold of the Cterminal domains may enhance its self-association as well as its interaction with the toxin, given that toxin-binding may induce the C-terminal domains to fold as well. As mentioned above, CopASO, at high concentration in the sample for NMR, associated and precipitated when titrated with the promoter DNA, thus detailed structural changes of the C-terminal domain caused by binding of the Nterminal domain to DNA was unable to be explored by NMR. Try of solving the structure of CopASO-DNA complex by X-Ray crystallography is ongoing. Author contributions XZ and XW conceived and supervised the study; RZ, XZ designed experiments; RZ, QL, and JZ performed experiments; JY provided materials, RZ, FL, LL and XZ analyzed data; RZ and XZ wrote the manuscript; LL and XW made manuscript revisions. Acknowledgements This work was supported by the National Natural Science Foundation of China [grant number 31470775]. Transparency document Transparency document related to this article can be found
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online at https://doi.org/10.1016/j.bbrc.2019.05.049. Appendix A. Supplementary data
[16]
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.049.
[17]
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