Backbone assignment of the N-terminal 24-kDa fragment of Escherichia coli topoisomerase IV ParE
subunit
Yan Li · Ying Lei Wong · Michelle Yueqi Lee · Hui Qi Ng · CongBao Kang
Y.Li · Y.L. Wong · M.Y.Lee · H.Q.Ng · C. Kang*
Experimental Therapeutics Centre, Agency for Science, Technology and Research, Singapore
138669
*Corresponding author
Email: [email protected]
31 Biopolis Way Nanos, #03-01, Singapore
Phone: 65-64070602
Fax: 65-64788768
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Abstract
Bacterial DNA topoisomerases are important drug targets due to their importance in DNA replication
and low homology to human topoisomerases. The N-terminal 24kDa region of E. coli topoisomerase IV E
subunit (eParE) contains the ATP binding pocket. Structure based drug discovery has been proven to be
an efficient way to develop potent ATP competitive inhibitors against ParEs. NMR spectroscopy is a
powerful tool to understand protein and inhibitor interactions in solution. In this study, we report the
backbone assignment for the N-terminal domain of E. coli ParE. The secondary structural information
and the assignment will aid in structure based antibacterial agents development targeting eParE.
Key words: NMR; topoisomerase; drug discovery; ParE; resonance assignment
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Biological content
Bacterial resistance to antibiotic agents has become a common problem in clinics. Therefore, it is
necessary to develop novel bacterial inhibitors that can overcome the drug resistance problems (Miller
and Waldrop 2010). To develop a novel bacterial agent, choosing a right target is one of the most
important steps in the drug discovery process. Accumulated studies have shown that targeting bacterial
topoisomerase is a feasible way to develop potent and novel antibacterial agents (Tari et al. 2013,
Basarab et al. 2013). Bacteria genome encodes two types of topoisomerases that are important for the
interconverstion of DNA topological isomers during DNA replication. Type I topoisomerase is responsible
for cleaving and rejoining one strand of DNA and type II topoisomerases play essential roles in cleaving
and rejoining a double strand DNA (Tse-Dinh 2009). Due to the low homology between human and
bacterial topoisomerases, several inhibitors targeting bacterial topoisomerases have been developed
(Basarab et al. 2013).
Bacterial type II topoisomerases contain two forms that are DNA gyrase and topoisomerase IV (Topo IV).
Functional DNA gyrase contains two subunits-Gyrase A and Gyrase B (GyrB) while functional Topo IV
contains C subunit (ParC) and E subunit (ParE). Both GyrB and ParE subunits contain an ATP binding
pocket at their N-termini that are approximately 24 kDa. The bacterial topoisomerase activities rely on
ATP, which makes the N-terminal ATP binding region of GyrB/ParE a suitable target to develop
inhibitors. X-ray structural studies have been conducted for both GyrB and ParE and the results show
that the folding of the N-terminal domains of these two proteins is very similar (Reece and Maxwell
1991, Wigley et al. 1991, Tsai et al. 1997, Bellon et al. 2004, Fu et al. 2009, Stanger, Dehio and Schirmer
2014). Fragment-based drug discovery has been shown to be an efficient way to develop ATP
competitive inhibitors targeting the N-terminal region of GyrB or ParE (Basarab et al. 2013). Several
potent compounds have been developed using this approach. For example, a potent compound with a
3
pyridylurea scaffold was designed using this approach (Basarab et al. 2013). Using structural information
of the GyrB and ParE, potent inhibitors such as tricyclic inhibitors have been developed (Tari et al. 2013).
Despite X-ray structural studies on GyrB and ParE, few NMR studies have been conducted for these
enzymes. NMR is a useful tool in study protein structure, protein-ligand interactions, and especially in
structure-based drug discovery. Understanding protein-inhibitor interaction in solution will make it
possible to develop broad-spectrum antibacterial agents. A backbone assignment of the N-terminal 24
kDa fragment of S. aureus and an assignment of the N-terminal 24 kDa fragment of E. coli GyrB are
available (Bellanda et al. 2002, Klaus et al. 2000), which was very useful for fragment-based drug
discovery (Chen et al. 2015). The free and ligand-bound form of N-terminal domain of pseudomonas
GyrB was also obtained, in which the drug binding sites were characterized (Li et al. 2015). In current
study, we report a backbone assignment for the free form of the N-terminal 24 kDa region of E. coli ParE
(eParE). The X-ray crystal structure of this region was determined (Bellon et al. 2004). Our assignment
together with the structural information of eParE will be helpful in structure based drug discovery
targeting bacterial DNA topoisomerases.
Methods and experiments
Protein sample preparation
The cDNA encoding the eParE was amplified by polymerase chain reaction using genomic DNA of E. coli
as a template. The resulting product was cloned into NdeI and XhoI sites of pET29b to generate a
plasmid-pET29-eParE that expresses residues 1-218 of ePaE and extra 8 residues (LEHHHHHH) at the Cterminus to aid in protein purification. The pET29-eParE was transformed in E. coli (BL21DE3) competent
cells. Several colonies from an LB plate were picked up and inoculated in 20 ml of M9 medium. The
overnight culture at 37 C was then transferred into 1 l of M9 medium that contained 1 g of 15NHCl4 and
2 g of
C-glucose. The recombinant protein was induced for 18 h at 18 C by adding β-D-1-
13
4
thiogalactopyranoside (IPTG) to 1 mM final concentration when the cell optical density (OD600) reached
0.6-1.0. The E. coli cells were harvested by centrifugation at 10, 000 ×g, 4 C and the cell pellet was
resuspended in a buffer that contained 20 mM sodium phosphate, 500 mM NaCl and 2 mM βmercaptoethanol. Cells were broken using a sonicator in an ice bath and the cell lysate were cleared by
centrifugation at 20, 000 ×g, 4 C for 20 min. The supernatant was mixed with Ni-NTA2+ resin and the
eParE was purified in a buffer that contained 500 mM imidazole, pH6.5. Protein was further purified
using gel filtration chromatography in which the sample was buffer exchanged to the NMR buffer that
contained 20 mM sodium phosphate, pH 7.2, 80 mM KCl, 2 mM DTT and 0.5 mM EDTA. A triple-labeled
sample (13C, 15N and 2H) was prepared by growing E. coli in a M9 medium that contained 1 g/l 15NH4Cl, 2
g/l 13C-glucose and D2O (99.9%).
NMR spectroscopy
The NMR experiments were carried out at 298K using Bruker Avance II 700 MHz equipped with a
cryoprobe. Uniformly 0.6 mM
15
N- and
13
C/15N/~70%2H- labeled proteins were used in NMR data
acquisition. Backbone assignments were obtained using two- (2D) and three-dimensional (3D)
experiments and transverse relaxation-optimized spectroscopy (TROSY) (Pervushin et al. 1998, Salzmann
et al. 1998)-based experiments including HSQC, HNCACB, HNCOCACB, HNCA, and HNCO. The proton
chemical shifts were referenced directly to DSS at 0 ppm and the
13
C and
15
N chemical shifts were
referenced indirectly to DSS using the absolute frequency ratios. All the spectra were processed using
NMRPipe (Delaglio et al. 1995) or Topspin 2.1 and analyzed using NMRView (Johnson 2004) and CARA
(http://www.mol.biol.ethz.ch/groups/wuthrich_group). The secondary structure was analyzed using
TALOS+ based on the backbone chemical shifts (Shen et al. 2009).
Assignment and data deposition
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The backbone resonance assignment of eParE was obtained using the conventional strategy based on
the 3D experiments. The cross peaks in the 1H-15N-TROSY spectrum were assigned and shown in Figure
1. The cross peaks of eParE are well dispersed in the spectrum, which is consistent with the fact that it
contains several β-strands. Most amide protons and amide resonances were assigned, except for
residues including M1, T2, Q3, N102, S108 and L111. Other backbone resonance assignments excluding
the 6 histidines at the C-terminus have nearly been completed. 98% of Cα (216 out of 220), 94% of Cβ
(188 out of 200) and 94% of C’ (206 of 220) have been obtained. The assignment has been deposited in
the in the BioMagResBank under accession number 26644.
Secondary structure of eParE
The secondary structural elements of eParE were identified using TALOS+ based on the obtained
chemical shifts. Free eParE contained 8 strands and 5 helices. The eight  strands include 1 (residues
53-59), 2 (residues 65-69), 3 (residues 125-131), 4 (residues 135-142), 5 (residues 150-152 and
156), 6 (residues 162-168), 7 (residues 198-204), and 8 (residues 210-215). The five α-helices include
α1 (residues 19-21), α2 (residues 33-48) α3 (residues 86-91), α4 (residues 116-120) and α5 (residues
181-194). Figure 2 shows the TALOS+ prediction as a function of residue number. Overall, the free eParE
in solution exhibits similar secondary structural elements to its novobiocin complex crystal structure.
Difference was also observed between X-ray and NMR structures (Fig. 2). X-ray structure shows that
there is a short helix formed by resides 19-21 at the N-terminus while NMR structural prediction shows
that there is short helix formed by residues 12-16 (Fig. 2). The difference observed may arise from the
fact that the N-terminal part of eParE is flexible. The available assignment will allow to map the inhibitor
binding sites on eParE.
Acknowledgments
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We appreciate financial support from A*STAR JCO grants (1331A028, 1231B015). We also thank Prof Ho
Sup Yoon and Dr. Hong Ye from Nanyang Technological University for the NMR experiments.
Figure legends
Figure 1 1H-15N-TROSY spectrum of a 13C/15N/2H-labeled N-terminal domain of eParE. The experiment
was conducted at 298K at a Bruker 700 MHz magnet. The protein was prepared in a buffer that
contained 20 mM sodium phosphate, pH7.2, 80 mM KCl, 2 mM DTT and 0.5 mM EDTA. The assigned
peaks are labeled with single-letter residue name and sequence number.
Figure 2. Secondary structure of eParE. TALOS+ index for secondary structure prediction are presented
by bars colored in red (helix) and blue (strand). The upper panel shows the secondary structure of eParE
derived from an X-ray structure (PDB id 1S14).
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