Characterization of the interaction between E. 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, Singapore, 138669

*Address corresponding to CongBao Kang: cbkang@etc.a-star.edu.sg;

Tel: +65-64070602

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Abstract

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 E. 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.

Key words: antibacterial agents; 19F NMR; drug design; docking; topoisomerase

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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 [6,7,8,9,10,11]. This has also led to the idea of

developing dual inhibitors of ParE and GyrB [12].

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 protein-inhibitor 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 protein-inhibitor complex will provide additional information

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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 bis-

pyridylurea inhibitor (inhibitor 1)- an ATP competitive inhibitor (Fig. 1). It binds to the ATP binding

pocket of ParE and inhibits both 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.

Materials and methods

Sample preparation and NMR measurement

Plasmid (pET29) was transformed into E. coli BL21DE 3 to express residues 1-218 of E. coli ParE and extra

8 residues (LEHHHHHH) at the C-terminus. Recombinant protein eParE was expressed and purified as

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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 700MHz 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+ based on the backbone chemical shifts [22].

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.

Protein-inhibitor interactions by NMR

To map the inhibitor binding site, 1H-15N-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 (Δ) were

calculated using the following equation. Δ=((ΔHN)2+(ΔN/5)2)0.5, where ΔHN is the chemical shift

changes upon inhibitor binding in the amide proton dimension and ΔN is the chemical shift changes in

the amide dimension [24]. To obtain protein-inhibitor inter-molecular NOEs, a NOESY-TROSY experiment

and a F1-13C/15N-filtered F2-15N-edited NOESY experiment with a mixing time of 120 ms was recorded

using a sample that contained 0.5 mM of 13C/15N /2H- labeled pGyrB and 1 mM of inhibitor. NOEs

observed in both spectra were considered as inter molecular NOEs.

Ligand-observed 1H and 19F 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, pH7.2, 80

mM KCl, 2 mM DTT and 0.5 mM EDTA. Purified eParE was prepared in the NMR buffer and aliquots were

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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.

IC50 assay for eParE

The ATP hydrolysis reactions were carried out in 30µl volumes containing the following mixture:

inhibitor 1 with the concentrations varying from 0.39 to 200 µM, 2% DMSO, 2 µM ParC, 2 µM ParE, 160

µM 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 hours and then quenched by

the addition of 30 µl of malachite green reagent containing 0.34 mg/ml of malachite green chloride and

0.011 g/ml of ammonium molybdate in 1M 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.

Results

Inhibitor 1 binds to eParE and inhibits its activity

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 of 0.51 M against eParE and MIC values of

more than 64 M against E. coli and 16 M 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 constructs used in the previous study [12].

This result was further corroborated with SPR studies and compound 1 was found to bind to eParE with

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KD 902 nM (Fig. 1C). Inhibitor 1 binds to eParE with a similar KD to ParE from Pseudomonas aeruginosa

and with a weaker affinity than E. coli GyrB and ParEs from Streptococcus pneumonia [23].

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 (Fig. 1, Fig. 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 (Fig. 1A, Fig. 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-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.

NMR study eParE-inhibitor 1 complex

To further understand the interactions between eParE and inhibitor 1, 1H-15N-HSQC experiments were

performed. NMR data collected on free eParE reveals well-dispersed cross peaks, representing a typical

spectrum for a β-barrel protein and its assignment has been obtained and 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

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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 TALOS+ based on the

assigned backbone chemical shifts (Fig. 3C). The result revealed that complex contains eight strands

and five α helices (Fig. 3C). The general secondary structure of eParE complex in solution is very similar

to its X-ray structures. However, the lengths of β1, β3, β5, β6, β7 and β8 differ in both X-ray and NMR

structures. Noteworthy is β5 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.

Chemical shift perturbation (CSP) caused by inhibitor binding

Inhibitor-induced CSP in the 1H-15N-HSQC spectrum has been widely used to map protein-inhibitor

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 Cα 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 α2, β2, β6, the loop between β2 and α3, α3 and α4 (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 Cα chemical

shifts are sensitive to the secondary structures. We found that the Cα atoms of several residues

including V39, N42 and E36 from α2, V87, I90 and L91 from α3, and R72, M74 and P75 from the loop

between β2 and α3 exhibited significant changes in the presence of inhibitors, suggesting that these

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residues are critical for inhibitor binding. The Cα 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 structure (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 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 Streptococcus 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 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.

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 structure-activity

relationship of the inhibitors [12]. The X-ray structures provide static conformations of protein-inhibitor

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 ligand-observed NMR spectroscopy, inhibitor

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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 (Fig. 1, Fig. 2). 19F NMR has been widely used in studying protein-

ligand interactions and ligand conformations due to its high sensitivity [30,31]. There is 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 sParE-inhibitor 1 complex is helpful to understand eParE and

inhibitor 1 interaction. 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 α3 and α4 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 in this region (Fig. 4A). On the other side, the loop between β2 and α3 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 model (Fig. 4), the residues from β 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 β 3 and 4 strands may improve inhibitor

potency.

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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 binding. Further docking and NOE

experiments demonstrated that inhibitor 1 binds to the ATP binding pocket.

Acknowledgments

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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Figure Legends

Figure 1. Biochemical and biophysical analysis of inhibitor 1 and eParE interaction. A. structure of

inhibitor 1. Numbers 1 and 2 are aliphatic carbons. B. IC50 curve of inhibitor 1 against eParE. C. SPR

analysis of inhibitor 1 and eParE interaction.

Figure 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.

Figure 3. Secondary structure of eParE and inhibitor complex. A. overlay of eParE in the absence (black)

and presence of inhibitor 1. B. Assignment of 1H-15N-HSQC of eParE-inhibitor 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.

Figure 4. Inhibitor 1 binds to ATP binding pocket of eParE. A. CSP caused by inhibitor 1 binding.

Δ=((ΔHN)2+(ΔN/5)2)0.5, where ΔHN is the chemical shift changes upon inhibitor binding in the amide

proton dimension and ΔN is the chemical shift changes in the amide dimension. B. Cα chemical shift

changes caused by inhibitor binding. ΔCα (Cα free-Cαcomplex) 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 ΔCα caused by inhibitor binding. Residues with ΔCα > 0.5 ppm, 0.2< ΔCα<0.5 are shown in red

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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.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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TOC

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