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Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor
Biochemical and Biophysical Research Communications xxx (2015) 1e6
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Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor Yan Li, Ying Lei Wong, Fui Mee Ng, Boping Liu, Yun Xuan Wong, Zhi Ying Poh, Siew Wen Then, Michelle Yueqi Lee, Hui Qi Ng, Alvin W. Hung, Joseph Cherian, Jeffrey Hill, Thomas H. Keller, CongBao Kang* Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos, #03-01, 138669, Singapore
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Article history: Received 6 October 2015 Accepted 7 October 2015 Available online xxx
Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE. © 2015 Elsevier Inc. All rights reserved.
Keywords: Antibacterial agents 19 F NMR Drug design Docking Topoisomerase
1. Introduction Bacterial topoisomerases play an important role in regulating DNA topological states [1]. Consequently, the DNA gyrase and topoisomerase IV are attractive drug targets because they are essential for bacterial DNA replication and share very low homology with eukaryotic topoisomerase [2]. Clinically approved antibacterial agents such as the fluoroquinolone class of inhibitors work via the interference of the bacterial topoisomerase enzyme [3]. The DNA gyrase is composed of A (GyrA) and B (GyrB) subunits and topoisomerase IV contains C (ParC) and E (ParE) subunits [4]. Fluoroquinolone binds to both DNA gyrase and topoisomerase IV and resistance have been shown to arise through mutations at it binding site [5]. Both GyrB and ParE contain an ATP binding pocket at their N-termini. The activities of bacterial topoisomerase depend on ATP, which makes the N-terminal ATP binding region of GyrB/ ParE an alternative site to develop inhibitors [4]. The natural product Novobiocin, has been shown to bind to the ATP binding pockets of both GyrB and ParE [6]. Structural studies have shown that the folding of the N-terminal domains of these two proteins is very similar [6e11]. This has also led to the idea of developing dual inhibitors of ParE and GyrB [12].
* Corresponding author. E-mail address: [email protected] (C. Kang).
Structure-based drug design (SBDD) is a powerful approach towards developing novel inhibitors against well characterized targets [13]. SBDD has been shown to play an important role in the development of antibacterial agents against ParE and GyrB [14]. With the aid of X-ray crystallography, nuclear magnet resonance (NMR) spectroscopy and homology modeling, several potent inhibitors of ParE and GyrB have been developed [12,14]. The understanding of proteineinhibitor interaction is very useful in the lead optimization phase of drug discovery. Studies have shown that many inhibitors bind tightly to GyrB/ParE with affinities at nanomolar range, while only few of them can inhibit bacterial growth [11,12,14]. Further biophysical characterization of the proteineinhibitor complex will provide additional information for lead optimization because crystallography analysis is providing most energetically stable conformation of the complex. Solution NMR spectroscopy is a useful tool in drug discovery because the changes of the chemical environments of residues in the absence and presence of an inhibitor can be monitored, which will be helpful for understanding the binding mode of the inhibitors under physiological conditions [15,16]. In this study, we carried out biochemical and biophysical studies to understand the molecular interactions between the N-terminal 24 kDa domain of Escherichia coli (E. coli) ParE (eParE) and a bispyridylurea inhibitor (inhibitor 1)- an ATP competitive inhibitor (Fig. 1). It binds to the ATP binding pocket of ParE and inhibits both
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Please cite this article in press as: Y. Li, et al., Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.036
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Y. Li et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6
ParE and GyrB activities [12,14]. Additionally, inhibitor 1 shows minimum inhibitory concentrations (MICs) against some bacteria. Although this inhibitor binds to ParE/GyrB ATP binding pocket, the effect of this inhibitor on the chemical environment of the residues from eParE is still unknown. Our previous study shows that chemical environment of several residues and thermal stability of GyrB of pseudomonas aeruginosa can be affected in the presence of inhibitor 1 [17]. Herein, we investigated the binding affinity between eParE and this inhibitor. We also investigated ligand conformation in solution using 19F NMR spectroscopy. Our results show that the IC50 of inhibitor 1 is 705 nM against eParE. The binding affinity is approximately 902 nM. Ligand-observed 19F NMR showed that ligand exists mainly in one conformation in solution and the interaction is undergoing slow exchange. Further NMR study and NOE experiments showed that inhibitor 1 binds to the ATP binding pocket of eParE. 2. Materials and methods 2.1. Sample preparation and NMR measurement Plasmid (pET29) was transformed into E. coli BL21DE 3 to express residues 1e218 of E. coli ParE and extra 8 residues (LEHHHHHH) at the C-terminus. Recombinant protein eParE was expressed and purified as previously described [17]. A sample containing 0.8 mM 13C/15N/2H-labeled eParE, 1.0 mM inhibitor 1, 20 mM sodium phosphate, pH 7.2, 80 mM KCl, 2 mM DTT and 0.5 mM EDTA was used for NMR data collection. NMR experiments were carried out on a Bruker AVANCE II 700 MHz magnet equipped with a cryoprobe. Transverse relaxation-optimized spectroscopy (TROSY) [18,19]-based 2D and 3D experiments including HSQC, HNCACB, HNCOCACB, HNCOCA, HNCA, HNCACO and HNCO were collected and processed. All the experiments were conducted at 25 C. All the spectra were processed using NMRPipe [20] or Topspin 2.1 and analyzed using NMRView [21] and CARA (http:// www.mol.biol.ethz.ch/groups/wuthrich_group). The secondary structure was analyzed using TALOS þ based on the backbone chemical shifts [22].
2.2. Surface Plasmon Resonance (SPR) measurement SPR experiments were carried out on a BIAcore-2000 system (GE Healthcare) at 25 C on CM5 chips. Purified protein was first immobilized on the chips as we previously described [23]. The buffer used in the binding study contained 10 mM Hepes, pH 7.5, 150 mM NaCl, 3 mM EDTA, and 0.005% v/v surfactant P20. The binding results were analyzed using the BIAcore T2000 Evaluation software (V2.0,GE Healthcare). Dissociation constant (KD) values were determined by the fitting of the data to 1:1 steady state binding model. 2.3. Proteineinhibitor interactions by NMR To map the inhibitor binding site, 1He15N-TROSY or HSQC spectra of pGyrB in the absence and presence of the inhibitor were collected and compared. Chemical shift perturbations (CSP) were monitored when inhibitor was added into the protein solution [24]. The combined chemical shift changes (Dd) were calculated using the following equation. Dd ¼ ((DdHN)2 þ (DdN/5)2)0.5, where DdHN is the chemical shift changes upon inhibitor binding in the amide proton dimension and DdN is the chemical shift changes in the amide dimension [24]. To obtain proteineinhibitor inter-molecular NOEs, a NOESY-TROSY experiment and a F1e13C/15N-filtered F2e15N-edited NOESY experiment with a mixing time of 120 ms was recorded using a sample that contained 0.5 mM of 13C/15N/2Hlabeled pGyrB and 1 mM of inhibitor. NOEs observed in both spectra were considered as inter molecular NOEs. 2.4. Ligand-observed 1H and
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F experiments
Ligand-observed NMR experiments were conducted on a Bruker 400 MHz magnet equipped with a BBO probe. Inhibitor 1 was prepared in an NMR buffer that contained 20 mM sodium phosphate, pH 7.2, 80 mM KCl, 2 mM DTT and 0.5 mM EDTA. Purified eParE was prepared in the NMR buffer and aliquots were made and lyophilized. 1H and 19F spectra of inhibitor 1 in the absence and presence of different amounts of eParE were recorded and processed using Topspin 2.1. 2.5. IC50 assay for eParE The ATP hydrolysis reactions were carried out in 30 ml volumes containing the following mixture: inhibitor 1 with the concentrations varying from 0.39 to 200 mM, 2% DMSO, 2 mM ParC, 2 mM ParE, 160 mM ATP, 20 mM Tris-HCL, pH8.0, 8 mM MgCl2,50 mM ammonium acetate, 2.5% (v/v) glycerol, 0.005% (v/v) Brij 35, 0.5 mM EDTA, 5 mM dithiothreitol, and 0.005 mg/ml salmon sperm DNA. The reactions were incubated at room temperature in a transparent 384-well plate for 24 h and then quenched by the addition of 30 ml of malachite green reagent containing 0.34 mg/ml of malachite green chloride and 0.011 g/ml of ammonium molybdate in 1 M HCl. After a 5-min incubation at room temperature, the absorbance at 650 nm (A650) was measured. A graph of A650 against log of compound concentration was plotted and the IC50 was determined as the inhibitor concentration giving 50% signal reduction. 3. Results 3.1. Inhibitor 1 binds to eParE and inhibits its activity
Fig. 1. Biochemical and biophysical analysis of inhibitor 1 and eParE interaction. A. structure of inhibitor 1. Numbers 1 and 2 are aliphatic carbons. F atoms are labeled with a circle. B. IC50 curve of inhibitor 1 against eParE. C. SPR analysis of inhibitor 1 and eParE interaction.
To develop potent ParE inhibitors, we first conducted biochemical and biophysical experiments to understand the interaction between ParE and a known inhibitor. We focused on E. coli ParE and inhibitor 1, a bis-pyridylurea scaffold that exhibits an IC50
Please cite this article in press as: Y. Li, et al., Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.036
Y. Li et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6
of 0.51 mM against eParE and MIC values of more than 64 mM against E. coli and 16 mM against E. coli tolC [12]. We first expressed and purified an N-terminal 24 kDa of E. coli ParE (eParE). Inhibitor 1 shows an IC50 of 705 nM against eParE (Fig. 1B). The higher IC50 obtained in this study may arise from the different construct from the previous study [12]. This result was further corroborated with SPR studies and compound 1 was found to bind to eParE with KD 902 nM (Fig. 1C). Inhibitor 1 binds to eParE with a similar KD to ParE from P. aeruginosa and with a weaker affinity than E. coli GyrB and ParEs from Streptococcus pneumoniae [23].
3.2. Inhibitor 1 binds to equal molar of eParE We used ligand-observed NMR spectroscopy to understand inhibitor 1 and eParE interactions. We first conducted 1H line broadening experiments for inhibitor 1 (Fig. 2A, B). The aliphatic proton signals are difficult to assign due to the buffer signals. The signals from the amide and aromatic protons of inhibitor 1 can be observed in the amide region (Figs. 1 and 2). Signal broadening was observed in the presence eParE, suggesting their interactions (Fig. 2A, B). The presence of protein and buffer signals makes the analysis difficult. In an effort to obtain better resolution in the ligand-based NMR experiments, 19F NMR spectrum was then acquired for inhibitor 1. There is only one peak observed for the free inhibitor because there is one CF3 group present in inhibitor 1 (Figs. 1A and 2C). In the presence of eParE, a new peak appears and the original peak decreases. When the amount of eParE was increased gradually, the newly appeared peak increased and the original peak decreased gradually, suggesting that the interaction is undergoing slow exchange in NMR timescale and indicating a high-
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affinity binding (Fig. 2). Additionally, the existence of only one broadened 19F peak for the complex, suggests that the inhibitor only exists in a single conformation in the complex. 3.3. NMR study eParE-inhibitor 1 complex To further understand the interactions between eParE and inhibitor 1, 1He15N-HSQC experiments were performed. NMR data collected on free eParE reveals well-dispersed cross peaks, representing a typical spectrum for a b-barrel protein and its assignment has been obtained and will be reported (Fig. 3A). In the presence of inhibitor 1, chemical shift perturbation observed (Fig. 3A). Varying the concentration of inhibitor in the experiments also resulted in a dose-depend manner, which is consistent with the 19F NMR result (Fig. 1C). Backbone resonance assignments for the complex were obtained (Fig. 2B). The assignments of the complex have been deposited in the biological magnetic Resonance Bank (BMRB) with accession number 26673. Secondary structures of eParE-inhibitor 1 complexes were predicted using TALOSþ based on the assigned backbone chemical shifts (Fig. 3C). The result revealed that complex contains eight b strands and five a helices (Fig. 3C). The general secondary structure of eParE complex in solution is very similar to its X-ray structures. However, the lengths of b1, b3, b5, b6, b7 and b8 differ in both X-ray and NMR structures. Noteworthy is b5 showed obvious difference in X-ray and NMR structures. This could be explained by the dynamic nature of this specific region of eParE. The residues forming the first and fourth helices are different (Fig. 3C), which again is caused by their dynamic natures because of their location [17]. More importantly, free and inhibitor-bound eParE exhibited almost identical secondary structures, suggesting that inhibitors did not cause significant secondary structural changes. 3.4. Chemical shift perturbation (CSP) caused by inhibitor binding
Fig. 2. Ligand-observed NMR analysis of inhibitor 1 and eParE interactions. A. 1H NMR of inhibitor 1 in the absence and presence of eParE. B. 1H NMR of inhibitor 1 in the amide proton region. Signal from inhibitor 1 is labeled with an asterisk. Protein to inhibitor ratio is shown and the appeared signals are from eParE. C. 19F NMR of inhibitor 1. Free inhibitor 1 signal is labeled with an asterisk. Protein to inhibitor ratio is labeled.
Inhibitor-induced CSP in the 1H-15N-HSQC spectrum has been widely used to map proteineinhibitor binding interface [25]. CSPs of eParE caused by binding to inhibitor 1 were obtained and plotted as a function of residue number (Fig. 4A). The changes of Ca chemical shifts in the absence and presence of inhibitor 1 are also plotted (Fig. 4B). Residues exhibited CSP in the 1H-15N-HSQC spectra upon inhibitor binding were mapped onto the X-ray structure of eParE (Fig. 4). Residues that exhibit large CSPs may contribute to inhibitor binding or affected by inhibitor binding. Predictably, most affected residues are those from the a2, b2, b6, the loop between b2 and a3, a3 and a4 (Fig. 4C), mirroring the results of Gyr B of P. ae and suggesting that inhibitor 1 bind to the ATP binding pocket of eParE. The Ca chemical shifts are sensitive to the secondary structures. We found that the Ca atoms of several residues including V39, N42 and E36 from a2, V87, I90 and L91 from a3, and R72, M74 and P75 from the loop between b2 and a3 exhibited significant changes in the presence of inhibitors, suggesting that these residues are critical for inhibitor binding. The Ca chemical shift changes in the presence of inhibitor 1 did not alter eParE secondary structural elements, which may arise from the fact that the free eParE is forming stable structures in solution (Fig. 3C). As CSP cannot provide the orientation of the ligand in the binding site [26], we carried out a docking study using high ambiguous restraints using HADDOCK [27,28]. A complex cluster was generated and the result suggested that inhibitor 1 binds to the ATP binding pocket. The co-crystal structure of inhibitor 1 and ParE (sParE) of S. pneumoniae (S. pn) was obtained [12]. The structure of sParE is very similar to eParE, which make it a reference to interpret our experimental results for eParE. Based on the model of the complex, the CSP induced by inhibitor binding, and the X-ray
Please cite this article in press as: Y. Li, et al., Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.036
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Fig. 3. Secondary structure of eParE and inhibitor complex. A. overlay of eParE in the absence (black) and presence (red) of inhibitor 1. B. Assignment of 1H15N-HSQC of eParEinhibitor 1 complex. The assigned peaks are labeled with residue name and sequence number. C. Secondary structure of eParE in complex with inhibitor 1. The secondary structure from the crystal structure (PDB id 1s14) is labeled as X-ray. Helices, loops, and strands are shown with boxes, lines and arrows, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
structure of sParE-inhibitor complex, we are confident that the orientation of inhibitor 1 in eParE is same as in sParE (Fig. 4D). Further 15N-edited and 15N-edited-13C/15N filtered NOEs experiment identified NOEs between the amide proton of V165 and inhibitor 1 (Fig. 4G), which further confirmed our model. 4. Discussion Bacterial topoisomerases have been proven to be a validated target for developing antibacterial agents [29]. SBDD has been used to develop potent inhibitors targeting ParE and GyrB [14]. X-ray structures of different GyrB/ParE-inhibitor complex provide useful information to understand structureeactivity relationship of the inhibitors [12]. The X-ray structures provide static conformations of proteineinhibitor complexes. Solution NMR and other biophysical study can provide additional information to understand the interaction in solution. Bacterial GyrBs and ParEs share high structural homology, and their interactions with available ATP competitive inhibitors have been well characterized using different methods [12,17]. However, there is no detailed study to understand and characterize conformational changes of the inhibitor in the complex with eParE. In this study, a potent pyridylurea inhibitor was used to understand protein and inhibitor interactions. Using ligandobserved NMR spectroscopy, inhibitor was confirmed to interact with eParE (Fig. 2). By using 19F spectroscopy, we found that the inhibitor 1 forms a tight complex with eParE because the interaction is in slow exchange, which is also consistent with its nano-molar binding affinity (Figs. 1 and 2). 19F NMR has been widely used in studying protein-ligand interactions and ligand conformations due to its high sensitivity [30,31]. There is a single 19F signal observed when inhibitor 1 forms complex with eParE, suggesting that inhibitor has only one conformation in the complex. In the presence of inhibitor 1, obvious CSP was observed for eParE (Fig. 3A). CSP was efficiently used to map compound binding
sites on a target protein (Fig. 3A). We confirmed that inhibitor 1 binds to the ATP binding pocket of eParE by performing CSP guided HADDOCK, comparing structure with sParE-inhibitor complex, and observing the inter-molecular NOEs between amide proton of V165 and inhibitor 1 (Fig. 4). The structural information of sParEinhibitor 1 complex is helpful to understand eParE and inhibitor 1 interactions. Based on the structural information obtained so far, it is evident that inhibitor 1 interacts with ParEs and GyrBs from E. coli, Pseudomonas and S. pn with nano-molar binding affinities and with similar binding site [12,17]. The loop region between a3 and a4 is flexible because there is no electron density map was observed in the X-ray structure and the residues with this loop exhibited broadened peaks in the spectrum (Fig. 3B). This loop is not involving in inhibitor binding because we did not observed CSP for residues within this region (Fig. 4A). On the other side, the loop between b2 and a3 is critical for inhibitor binding. Significant CSP was observed for residues in this region (Fig. 4B). One of the most important residues is Met 74 and a study showed that mutation of this residue to others can change the compound binding affinity [6] (Fig. 4). This loop acts as a cover on the binding pocket. Based on the structures and the complex model (Fig. 4), the residues from b strands 3 and 4 are not affected by inhibitor binding. This may arise from the presence of the loop that separate inhibitor from the two strands. Further compound optimization to gain interaction with the b3 and 4 strands may improve inhibitor potency. In summary, we conducted biochemical and biophysical characterization of the interaction between eParE and inhibitor 1 in this study. Inhibitor 1 was able to inhibit the enzymatic activity of the N-terminal 24 kDa region of E. coli ParE, suggesting that this construct can be used in biochemical assay to evaluate potential drug candidates. Ligand-observed 19F NMR was applied to understand eParE and inhibitor 1 interaction and this inhibitor was confirmed to have one conformation in the complex. Using chemical shift perturbation, we identified important residues for ligand
Please cite this article in press as: Y. Li, et al., Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.036
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Fig. 4. Inhibitor 1 binds to ATP binding pocket of eParE. A. CSP caused by inhibitor 1 binding. Dd ¼ ((DdHN)2 þ (DdN/5)2)0.5, where DdHN is the chemical shift changes upon inhibitor binding in the amide proton dimension and DdN is the chemical shift changes in the amide dimension. B. Ca chemical shift changes caused by inhibitor binding. DCa (CafreeCacomplex) is plotted against residue number. The assignment for free eParE is submitted elsewhere (BMRB 26644). C. Structure of eParE. Left panel is the X-ray structure of eParE (PDB id 1S14). Novobiocin is shown in pink. The secondary elements are labeled. The middle panel is the CSP caused by inhibitor binding. Residues with CSP >0.3 ppm, 0.2 < CSP < 0.3 ppm, and 0.1 < CSP < 0.2 are shown in red, light blue, and dark blue, respectively. The right panel is DCa caused by inhibitor binding. Residues with DCa > 0.5 ppm, 0.2 < DCa < 0.5 are shown in red and blue, respectively. D. HADDOCK of eParE and inhibitor 1 complex. Overlay of several models of the complex using HADDOCK based on the CSP observed. The inhibitors are shown in sticks. E. One model showing similar structure to the inhibitor 1-sParE complex (F, PDB id 4LP0). G. NOEs observed between V165 and inhibitor 1. Left panel is the slice of V165 in the NOESY-TROSY spectrum. Right panel is the slice from a filtered NOESY experiment. The signals from inhibitor 1 are labeled with dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
binding. Further docking and NOE experiments demonstrated that inhibitor 1 binds to the ATP binding pocket. Conflict of interest The authors declare there is no conflict of interest.
[4] [5] [6]
Acknowledgments [7]
We appreciate the financial support from A*STAR JCO grants (1331A028, 1231B015). The authors appreciate valuable discussion and suggestion from members of the drug discovery team in ETC, A*STAR.
[8]
[9]
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Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor Yan Li, Ying Lei Wong, Fui Mee Ng, Boping Liu, Yun Xuan Wong, Zhi Ying Poh, Siew Wen Then, Michelle Yueqi Lee, Hui Qi Ng, Alvin W. Hung, Joseph Cherian, Jeffrey Hill, Thomas H. Keller, CongBao Kang* Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos, #03-01, 138669, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 October 2015 Accepted 7 October 2015 Available online xxx
Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE. © 2015 Elsevier Inc. All rights reserved.
Keywords: Antibacterial agents 19 F NMR Drug design Docking Topoisomerase
1. Introduction Bacterial topoisomerases play an important role in regulating DNA topological states [1]. Consequently, the DNA gyrase and topoisomerase IV are attractive drug targets because they are essential for bacterial DNA replication and share very low homology with eukaryotic topoisomerase [2]. Clinically approved antibacterial agents such as the fluoroquinolone class of inhibitors work via the interference of the bacterial topoisomerase enzyme [3]. The DNA gyrase is composed of A (GyrA) and B (GyrB) subunits and topoisomerase IV contains C (ParC) and E (ParE) subunits [4]. Fluoroquinolone binds to both DNA gyrase and topoisomerase IV and resistance have been shown to arise through mutations at it binding site [5]. Both GyrB and ParE contain an ATP binding pocket at their N-termini. The activities of bacterial topoisomerase depend on ATP, which makes the N-terminal ATP binding region of GyrB/ ParE an alternative site to develop inhibitors [4]. The natural product Novobiocin, has been shown to bind to the ATP binding pockets of both GyrB and ParE [6]. Structural studies have shown that the folding of the N-terminal domains of these two proteins is very similar [6e11]. This has also led to the idea of developing dual inhibitors of ParE and GyrB [12].
* Corresponding author. E-mail address: [email protected] (C. Kang).
Structure-based drug design (SBDD) is a powerful approach towards developing novel inhibitors against well characterized targets [13]. SBDD has been shown to play an important role in the development of antibacterial agents against ParE and GyrB [14]. With the aid of X-ray crystallography, nuclear magnet resonance (NMR) spectroscopy and homology modeling, several potent inhibitors of ParE and GyrB have been developed [12,14]. The understanding of proteineinhibitor interaction is very useful in the lead optimization phase of drug discovery. Studies have shown that many inhibitors bind tightly to GyrB/ParE with affinities at nanomolar range, while only few of them can inhibit bacterial growth [11,12,14]. Further biophysical characterization of the proteineinhibitor complex will provide additional information for lead optimization because crystallography analysis is providing most energetically stable conformation of the complex. Solution NMR spectroscopy is a useful tool in drug discovery because the changes of the chemical environments of residues in the absence and presence of an inhibitor can be monitored, which will be helpful for understanding the binding mode of the inhibitors under physiological conditions [15,16]. In this study, we carried out biochemical and biophysical studies to understand the molecular interactions between the N-terminal 24 kDa domain of Escherichia coli (E. coli) ParE (eParE) and a bispyridylurea inhibitor (inhibitor 1)- an ATP competitive inhibitor (Fig. 1). It binds to the ATP binding pocket of ParE and inhibits both
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Please cite this article in press as: Y. Li, et al., Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.036
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ParE and GyrB activities [12,14]. Additionally, inhibitor 1 shows minimum inhibitory concentrations (MICs) against some bacteria. Although this inhibitor binds to ParE/GyrB ATP binding pocket, the effect of this inhibitor on the chemical environment of the residues from eParE is still unknown. Our previous study shows that chemical environment of several residues and thermal stability of GyrB of pseudomonas aeruginosa can be affected in the presence of inhibitor 1 [17]. Herein, we investigated the binding affinity between eParE and this inhibitor. We also investigated ligand conformation in solution using 19F NMR spectroscopy. Our results show that the IC50 of inhibitor 1 is 705 nM against eParE. The binding affinity is approximately 902 nM. Ligand-observed 19F NMR showed that ligand exists mainly in one conformation in solution and the interaction is undergoing slow exchange. Further NMR study and NOE experiments showed that inhibitor 1 binds to the ATP binding pocket of eParE. 2. Materials and methods 2.1. Sample preparation and NMR measurement Plasmid (pET29) was transformed into E. coli BL21DE 3 to express residues 1e218 of E. coli ParE and extra 8 residues (LEHHHHHH) at the C-terminus. Recombinant protein eParE was expressed and purified as previously described [17]. A sample containing 0.8 mM 13C/15N/2H-labeled eParE, 1.0 mM inhibitor 1, 20 mM sodium phosphate, pH 7.2, 80 mM KCl, 2 mM DTT and 0.5 mM EDTA was used for NMR data collection. NMR experiments were carried out on a Bruker AVANCE II 700 MHz magnet equipped with a cryoprobe. Transverse relaxation-optimized spectroscopy (TROSY) [18,19]-based 2D and 3D experiments including HSQC, HNCACB, HNCOCACB, HNCOCA, HNCA, HNCACO and HNCO were collected and processed. All the experiments were conducted at 25 C. All the spectra were processed using NMRPipe [20] or Topspin 2.1 and analyzed using NMRView [21] and CARA (http:// www.mol.biol.ethz.ch/groups/wuthrich_group). The secondary structure was analyzed using TALOS þ based on the backbone chemical shifts [22].
2.2. Surface Plasmon Resonance (SPR) measurement SPR experiments were carried out on a BIAcore-2000 system (GE Healthcare) at 25 C on CM5 chips. Purified protein was first immobilized on the chips as we previously described [23]. The buffer used in the binding study contained 10 mM Hepes, pH 7.5, 150 mM NaCl, 3 mM EDTA, and 0.005% v/v surfactant P20. The binding results were analyzed using the BIAcore T2000 Evaluation software (V2.0,GE Healthcare). Dissociation constant (KD) values were determined by the fitting of the data to 1:1 steady state binding model. 2.3. Proteineinhibitor interactions by NMR To map the inhibitor binding site, 1He15N-TROSY or HSQC spectra of pGyrB in the absence and presence of the inhibitor were collected and compared. Chemical shift perturbations (CSP) were monitored when inhibitor was added into the protein solution [24]. The combined chemical shift changes (Dd) were calculated using the following equation. Dd ¼ ((DdHN)2 þ (DdN/5)2)0.5, where DdHN is the chemical shift changes upon inhibitor binding in the amide proton dimension and DdN is the chemical shift changes in the amide dimension [24]. To obtain proteineinhibitor inter-molecular NOEs, a NOESY-TROSY experiment and a F1e13C/15N-filtered F2e15N-edited NOESY experiment with a mixing time of 120 ms was recorded using a sample that contained 0.5 mM of 13C/15N/2Hlabeled pGyrB and 1 mM of inhibitor. NOEs observed in both spectra were considered as inter molecular NOEs. 2.4. Ligand-observed 1H and
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F experiments
Ligand-observed NMR experiments were conducted on a Bruker 400 MHz magnet equipped with a BBO probe. Inhibitor 1 was prepared in an NMR buffer that contained 20 mM sodium phosphate, pH 7.2, 80 mM KCl, 2 mM DTT and 0.5 mM EDTA. Purified eParE was prepared in the NMR buffer and aliquots were made and lyophilized. 1H and 19F spectra of inhibitor 1 in the absence and presence of different amounts of eParE were recorded and processed using Topspin 2.1. 2.5. IC50 assay for eParE The ATP hydrolysis reactions were carried out in 30 ml volumes containing the following mixture: inhibitor 1 with the concentrations varying from 0.39 to 200 mM, 2% DMSO, 2 mM ParC, 2 mM ParE, 160 mM ATP, 20 mM Tris-HCL, pH8.0, 8 mM MgCl2,50 mM ammonium acetate, 2.5% (v/v) glycerol, 0.005% (v/v) Brij 35, 0.5 mM EDTA, 5 mM dithiothreitol, and 0.005 mg/ml salmon sperm DNA. The reactions were incubated at room temperature in a transparent 384-well plate for 24 h and then quenched by the addition of 30 ml of malachite green reagent containing 0.34 mg/ml of malachite green chloride and 0.011 g/ml of ammonium molybdate in 1 M HCl. After a 5-min incubation at room temperature, the absorbance at 650 nm (A650) was measured. A graph of A650 against log of compound concentration was plotted and the IC50 was determined as the inhibitor concentration giving 50% signal reduction. 3. Results 3.1. Inhibitor 1 binds to eParE and inhibits its activity
Fig. 1. Biochemical and biophysical analysis of inhibitor 1 and eParE interaction. A. structure of inhibitor 1. Numbers 1 and 2 are aliphatic carbons. F atoms are labeled with a circle. B. IC50 curve of inhibitor 1 against eParE. C. SPR analysis of inhibitor 1 and eParE interaction.
To develop potent ParE inhibitors, we first conducted biochemical and biophysical experiments to understand the interaction between ParE and a known inhibitor. We focused on E. coli ParE and inhibitor 1, a bis-pyridylurea scaffold that exhibits an IC50
Please cite this article in press as: Y. Li, et al., Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.036
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of 0.51 mM against eParE and MIC values of more than 64 mM against E. coli and 16 mM against E. coli tolC [12]. We first expressed and purified an N-terminal 24 kDa of E. coli ParE (eParE). Inhibitor 1 shows an IC50 of 705 nM against eParE (Fig. 1B). The higher IC50 obtained in this study may arise from the different construct from the previous study [12]. This result was further corroborated with SPR studies and compound 1 was found to bind to eParE with KD 902 nM (Fig. 1C). Inhibitor 1 binds to eParE with a similar KD to ParE from P. aeruginosa and with a weaker affinity than E. coli GyrB and ParEs from Streptococcus pneumoniae [23].
3.2. Inhibitor 1 binds to equal molar of eParE We used ligand-observed NMR spectroscopy to understand inhibitor 1 and eParE interactions. We first conducted 1H line broadening experiments for inhibitor 1 (Fig. 2A, B). The aliphatic proton signals are difficult to assign due to the buffer signals. The signals from the amide and aromatic protons of inhibitor 1 can be observed in the amide region (Figs. 1 and 2). Signal broadening was observed in the presence eParE, suggesting their interactions (Fig. 2A, B). The presence of protein and buffer signals makes the analysis difficult. In an effort to obtain better resolution in the ligand-based NMR experiments, 19F NMR spectrum was then acquired for inhibitor 1. There is only one peak observed for the free inhibitor because there is one CF3 group present in inhibitor 1 (Figs. 1A and 2C). In the presence of eParE, a new peak appears and the original peak decreases. When the amount of eParE was increased gradually, the newly appeared peak increased and the original peak decreased gradually, suggesting that the interaction is undergoing slow exchange in NMR timescale and indicating a high-
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affinity binding (Fig. 2). Additionally, the existence of only one broadened 19F peak for the complex, suggests that the inhibitor only exists in a single conformation in the complex. 3.3. NMR study eParE-inhibitor 1 complex To further understand the interactions between eParE and inhibitor 1, 1He15N-HSQC experiments were performed. NMR data collected on free eParE reveals well-dispersed cross peaks, representing a typical spectrum for a b-barrel protein and its assignment has been obtained and will be reported (Fig. 3A). In the presence of inhibitor 1, chemical shift perturbation observed (Fig. 3A). Varying the concentration of inhibitor in the experiments also resulted in a dose-depend manner, which is consistent with the 19F NMR result (Fig. 1C). Backbone resonance assignments for the complex were obtained (Fig. 2B). The assignments of the complex have been deposited in the biological magnetic Resonance Bank (BMRB) with accession number 26673. Secondary structures of eParE-inhibitor 1 complexes were predicted using TALOSþ based on the assigned backbone chemical shifts (Fig. 3C). The result revealed that complex contains eight b strands and five a helices (Fig. 3C). The general secondary structure of eParE complex in solution is very similar to its X-ray structures. However, the lengths of b1, b3, b5, b6, b7 and b8 differ in both X-ray and NMR structures. Noteworthy is b5 showed obvious difference in X-ray and NMR structures. This could be explained by the dynamic nature of this specific region of eParE. The residues forming the first and fourth helices are different (Fig. 3C), which again is caused by their dynamic natures because of their location [17]. More importantly, free and inhibitor-bound eParE exhibited almost identical secondary structures, suggesting that inhibitors did not cause significant secondary structural changes. 3.4. Chemical shift perturbation (CSP) caused by inhibitor binding
Fig. 2. Ligand-observed NMR analysis of inhibitor 1 and eParE interactions. A. 1H NMR of inhibitor 1 in the absence and presence of eParE. B. 1H NMR of inhibitor 1 in the amide proton region. Signal from inhibitor 1 is labeled with an asterisk. Protein to inhibitor ratio is shown and the appeared signals are from eParE. C. 19F NMR of inhibitor 1. Free inhibitor 1 signal is labeled with an asterisk. Protein to inhibitor ratio is labeled.
Inhibitor-induced CSP in the 1H-15N-HSQC spectrum has been widely used to map proteineinhibitor binding interface [25]. CSPs of eParE caused by binding to inhibitor 1 were obtained and plotted as a function of residue number (Fig. 4A). The changes of Ca chemical shifts in the absence and presence of inhibitor 1 are also plotted (Fig. 4B). Residues exhibited CSP in the 1H-15N-HSQC spectra upon inhibitor binding were mapped onto the X-ray structure of eParE (Fig. 4). Residues that exhibit large CSPs may contribute to inhibitor binding or affected by inhibitor binding. Predictably, most affected residues are those from the a2, b2, b6, the loop between b2 and a3, a3 and a4 (Fig. 4C), mirroring the results of Gyr B of P. ae and suggesting that inhibitor 1 bind to the ATP binding pocket of eParE. The Ca chemical shifts are sensitive to the secondary structures. We found that the Ca atoms of several residues including V39, N42 and E36 from a2, V87, I90 and L91 from a3, and R72, M74 and P75 from the loop between b2 and a3 exhibited significant changes in the presence of inhibitors, suggesting that these residues are critical for inhibitor binding. The Ca chemical shift changes in the presence of inhibitor 1 did not alter eParE secondary structural elements, which may arise from the fact that the free eParE is forming stable structures in solution (Fig. 3C). As CSP cannot provide the orientation of the ligand in the binding site [26], we carried out a docking study using high ambiguous restraints using HADDOCK [27,28]. A complex cluster was generated and the result suggested that inhibitor 1 binds to the ATP binding pocket. The co-crystal structure of inhibitor 1 and ParE (sParE) of S. pneumoniae (S. pn) was obtained [12]. The structure of sParE is very similar to eParE, which make it a reference to interpret our experimental results for eParE. Based on the model of the complex, the CSP induced by inhibitor binding, and the X-ray
Please cite this article in press as: Y. Li, et al., Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.036
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Fig. 3. Secondary structure of eParE and inhibitor complex. A. overlay of eParE in the absence (black) and presence (red) of inhibitor 1. B. Assignment of 1H15N-HSQC of eParEinhibitor 1 complex. The assigned peaks are labeled with residue name and sequence number. C. Secondary structure of eParE in complex with inhibitor 1. The secondary structure from the crystal structure (PDB id 1s14) is labeled as X-ray. Helices, loops, and strands are shown with boxes, lines and arrows, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
structure of sParE-inhibitor complex, we are confident that the orientation of inhibitor 1 in eParE is same as in sParE (Fig. 4D). Further 15N-edited and 15N-edited-13C/15N filtered NOEs experiment identified NOEs between the amide proton of V165 and inhibitor 1 (Fig. 4G), which further confirmed our model. 4. Discussion Bacterial topoisomerases have been proven to be a validated target for developing antibacterial agents [29]. SBDD has been used to develop potent inhibitors targeting ParE and GyrB [14]. X-ray structures of different GyrB/ParE-inhibitor complex provide useful information to understand structureeactivity relationship of the inhibitors [12]. The X-ray structures provide static conformations of proteineinhibitor complexes. Solution NMR and other biophysical study can provide additional information to understand the interaction in solution. Bacterial GyrBs and ParEs share high structural homology, and their interactions with available ATP competitive inhibitors have been well characterized using different methods [12,17]. However, there is no detailed study to understand and characterize conformational changes of the inhibitor in the complex with eParE. In this study, a potent pyridylurea inhibitor was used to understand protein and inhibitor interactions. Using ligandobserved NMR spectroscopy, inhibitor was confirmed to interact with eParE (Fig. 2). By using 19F spectroscopy, we found that the inhibitor 1 forms a tight complex with eParE because the interaction is in slow exchange, which is also consistent with its nano-molar binding affinity (Figs. 1 and 2). 19F NMR has been widely used in studying protein-ligand interactions and ligand conformations due to its high sensitivity [30,31]. There is a single 19F signal observed when inhibitor 1 forms complex with eParE, suggesting that inhibitor has only one conformation in the complex. In the presence of inhibitor 1, obvious CSP was observed for eParE (Fig. 3A). CSP was efficiently used to map compound binding
sites on a target protein (Fig. 3A). We confirmed that inhibitor 1 binds to the ATP binding pocket of eParE by performing CSP guided HADDOCK, comparing structure with sParE-inhibitor complex, and observing the inter-molecular NOEs between amide proton of V165 and inhibitor 1 (Fig. 4). The structural information of sParEinhibitor 1 complex is helpful to understand eParE and inhibitor 1 interactions. Based on the structural information obtained so far, it is evident that inhibitor 1 interacts with ParEs and GyrBs from E. coli, Pseudomonas and S. pn with nano-molar binding affinities and with similar binding site [12,17]. The loop region between a3 and a4 is flexible because there is no electron density map was observed in the X-ray structure and the residues with this loop exhibited broadened peaks in the spectrum (Fig. 3B). This loop is not involving in inhibitor binding because we did not observed CSP for residues within this region (Fig. 4A). On the other side, the loop between b2 and a3 is critical for inhibitor binding. Significant CSP was observed for residues in this region (Fig. 4B). One of the most important residues is Met 74 and a study showed that mutation of this residue to others can change the compound binding affinity [6] (Fig. 4). This loop acts as a cover on the binding pocket. Based on the structures and the complex model (Fig. 4), the residues from b strands 3 and 4 are not affected by inhibitor binding. This may arise from the presence of the loop that separate inhibitor from the two strands. Further compound optimization to gain interaction with the b3 and 4 strands may improve inhibitor potency. In summary, we conducted biochemical and biophysical characterization of the interaction between eParE and inhibitor 1 in this study. Inhibitor 1 was able to inhibit the enzymatic activity of the N-terminal 24 kDa region of E. coli ParE, suggesting that this construct can be used in biochemical assay to evaluate potential drug candidates. Ligand-observed 19F NMR was applied to understand eParE and inhibitor 1 interaction and this inhibitor was confirmed to have one conformation in the complex. Using chemical shift perturbation, we identified important residues for ligand
Please cite this article in press as: Y. Li, et al., Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.036
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Fig. 4. Inhibitor 1 binds to ATP binding pocket of eParE. A. CSP caused by inhibitor 1 binding. Dd ¼ ((DdHN)2 þ (DdN/5)2)0.5, where DdHN is the chemical shift changes upon inhibitor binding in the amide proton dimension and DdN is the chemical shift changes in the amide dimension. B. Ca chemical shift changes caused by inhibitor binding. DCa (CafreeCacomplex) is plotted against residue number. The assignment for free eParE is submitted elsewhere (BMRB 26644). C. Structure of eParE. Left panel is the X-ray structure of eParE (PDB id 1S14). Novobiocin is shown in pink. The secondary elements are labeled. The middle panel is the CSP caused by inhibitor binding. Residues with CSP >0.3 ppm, 0.2 < CSP < 0.3 ppm, and 0.1 < CSP < 0.2 are shown in red, light blue, and dark blue, respectively. The right panel is DCa caused by inhibitor binding. Residues with DCa > 0.5 ppm, 0.2 < DCa < 0.5 are shown in red and blue, respectively. D. HADDOCK of eParE and inhibitor 1 complex. Overlay of several models of the complex using HADDOCK based on the CSP observed. The inhibitors are shown in sticks. E. One model showing similar structure to the inhibitor 1-sParE complex (F, PDB id 4LP0). G. NOEs observed between V165 and inhibitor 1. Left panel is the slice of V165 in the NOESY-TROSY spectrum. Right panel is the slice from a filtered NOESY experiment. The signals from inhibitor 1 are labeled with dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
binding. Further docking and NOE experiments demonstrated that inhibitor 1 binds to the ATP binding pocket. Conflict of interest The authors declare there is no conflict of interest.
[4] [5] [6]
Acknowledgments [7]
We appreciate the financial support from A*STAR JCO grants (1331A028, 1231B015). The authors appreciate valuable discussion and suggestion from members of the drug discovery team in ETC, A*STAR.
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Please cite this article in press as: Y. Li, et al., Characterization of the interaction between Escherichia coli topoisomerase IV E subunit and an ATP competitive inhibitor, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.036