NMR structural characterization of the N-terminal active domain of the gyrase B subunit from
Pseudomonas aeruginosa and its complex with an inhibitor
Yan Li, Yun Xuan Wong, Zhi Ying Poh, Ying Lei Wong, Michelle Yueqi Lee, Hui Qi Ng, Boping Liu, 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: [email protected];
Tel: +65-64070602
1
Abstract
The N-terminal ATP binding domain of the DNA gyrase B subunit is a validated drug target for
antibacterial drug discovery. Structural information for this domain (pGyrB) from Pseudomonas
aeruginosa (P. ae) is still missing. In this study, the interaction between pGyrB and a bis-pyridylurea
inhibitor was characterized using several biophysical methods. We further carried out structural analysis
of pGyrB using NMR spectroscopy. The secondary structures of free and inhibitor bound pGyrB were
obtained based on backbone chemical shift assignment. Chemical shift perturbation and NOE
experiments demonstrated that the inhibitor binds to the ATP binding pocket. The results of this study
will be helpful for drug development targeting P. ae.
Keywords: NMR; drug discovery; topoisomerase; gyrase B; structure-based drug design
2
1. Introduction
The bacterial genome encodes two types of topoisomerases, I and II which differ in the mechanism of
DNA strand breakage [1]. The type II topoisomerases consist of two types, DNA gyrase and
topoisomerase IV (TopoIV). These enzymes play essential roles in DNA replication by managing the
topological states of DNA in the cell [2,3]. In prokaryotes, type II topoisomerases consist of two subunits
and are functional in tetrameric form, which is different from the eukaryotic type II topoisomerases that
exist as homodimers [3]. Prokaryotic DNA gyrase contains two gyrase A (GyrA) and gyrase B (GyrB)
subunits respectively to form the heterotetramer. For Escherichia coli (E. coli), GyrA is a 97 kDa protein
that is involved in DNA binding and GyrB, it is a 90 kDa protein with an N-terminal ATP binding domain
[4].
Interfering with bacterial DNA replication by targeting type II topoisomerases has been shown to be an
efficient strategy to develop antibacterial agents [5]. Successful examples include the fluoroquinolone
class of antibiotics [6]. Many other novel and potent inhibitors have been developed in recent years [7].
The N-terminal domain of the type II topoisomerases contains the ATP binding pocket and has been of
great interest in drug development because this domain exhibited high sequence homology among
pathogenic bacteria and low homology with eukaryotes [5,7]. Structure-based drug design has been
demonstrated to be a powerful tool in developing inhibitors targeting both GyrB and TopolV ATP binding
domains. Several classes of inhibitors have been discovered using this approach [8-11].
The structures of the N-terminal ATP binding domains of both GyrB and E subunit of TopoIV (ParE) from
E. coli have been reported [12,13]. The structures of GyrB/ParE and inhibitor complexes demonstrated
that most of the inhibitors are binding with the ATP binding pocket [9]. Although the structure of this
domain is similar among the type II topoisomerases, a single residue difference among different
topoisomerases can result in different inhibitor potency [2]. Understanding protein-inhibitor
interactions will provide useful information in the drug development process. NMR spectroscopy has
been proven to be a useful tool in drug development [14]. Despite the extensive X-ray studies of GyrBs
and ParEs, few NMR studies have been conducted for the N-terminal domain of GyrB and ParE from
bacteria except for the assignments of the P24 fragment of S. aureus and the N-terminal 24 kDa
fragment of GyrB from E. coli [15,16].
In this study, we obtained the N-terminal 24 kDa domain of the GyrB from Pseudomonas aeruginosa (P.
ae) (referred as pGyrB) for NMR studies. As the structure of this domain is not available, structural
3
information for this domain will be useful for structure-based drug design because P. ae is an important
pathogenic species. We managed to obtain the backbone assignments for both free and inhibitor-bound
forms of pGyrB. The secondary structure and dynamic property of pGyrB in solution were analyzed and
the bis-pyridylurea inhibitor was shown to bind to the ATP binding pocket.
2. Materials and Methods
2.1 Sample preparation
The cDNA encoding the pGyrB was amplified by polymerase chain reaction using genomic DNA of P. ae
as a template and cloned into NdeI and XhoI sites of pET29b. The resulting plasmid can express residues
1-222 of GyrB and extra 8 residues (LEHHHHHH) at the C-terminus. To express pGyrB from E. coli, the
plasmid was transformed in E. coli (BL21DE3) competent cells. The recombinant protein was expressed
and purified using affinity purification and gel filtration chromatography [17,18]. Briefly, several colonies
were picked up from the plate and inoculated in 20 ml of M9 medium. The overnight culture at 37 C
was then transferred into 1 l of M9 medium. The recombinant protein was induced for 18 h at 18 C by
adding β-D-1-thiogalactopyranoside (IPTG) to 1 mM. The E. coli cells were harvested by centrifugation
and the recombinant protein was purified in a buffer that contained 20 mM sodium phosphate, pH 6.5,
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
15
NH4Cl, 2 g/l 2H-13C-glucose and D2O (99.9%).
Purified protein was concentrated to 0.5-0.8 mM for further studies.
2.2 Backbone resonance assignment
Uniformly 15N- and 13C/15N/2H- labeled proteins were used in NMR data acquisition. Two- (2D) and threedimensional (3D) experiments and transverse relaxation-optimized spectroscopy (TROSY) [19,20]-based
experiments including HSQC, HNCACB, HNCOCACB, HNCOCA, HNCA, HNCOCA, HNCACO and HNCO were
collected and processed. For pGyrB and inhibitor complex, protein was first purified and inhibitor was
then added into the solution to a protein: inhibitor molar ratio of 1:1.2. Inhibitor was synthesized and
purified as described [21]. All the experiments were conducted at 25 C on a Bruker Avance 700
spectrometer equipped with a cryoprobe. All the spectra were processed using NMRPipe [22] or Topspin
2.1 and analyzed using NMRView [23] and CARA (http://www.mol.biol.ethz.ch/groups/wuthrich_group).
The secondary structure was analyzed using TALOS+ based on the backbone chemical shifts [24].
4
2.3 Protein-inhibitor interactions
1
H-15N-HSQC spectra of pGyrB in the absence and presence of the inhibitor were compared and chemical
shift perturbations (CSP) were monitored [25]. The combined chemical shift changes (Δ) were
calculated using the following equation. Δ=((ΔHN)2+(ΔN/5)2)0.5, where ΔHN is the chemical shift
changes upon inhibitor binding in the amide proton dimension and ΔN is the chemical shift changes in
the amide dimension [25]. To obtain protein-inhibitor inter-molecular NOEs, a NOESY-TROSY experiment
with a mixing time of 100 ms was recorded using a sample that contained 0.5 mM of 13C/15N /2H- labeled
pGyrB and 1 mM of inhibitor.
2.4 Effect of inhibitor on protein thermal stability
Thermal shift experiment was carried out on a Roche LC480 PCR machine. Each assay well contained 10
M pGyrB, 20 × spyro orange. The assay buffer contained 50 mM HEPES, pH7.2, 250 mM NaCl and 5 mM
MgCl2.
2.5 Isothermal Titration Calorimetry (ITC) experiment
ITC experiment was performed on an Auto-iTC200 instrument (Microcal Inc.). The experiment was
carried out at 25 C. Protein was prepared in a buffer that contained 50 mM HEPES, pH7.5, 250 mM
NaCl and 5 mM MgCl2 at concentrations of 100 M. Inhibitor was prepared in the same buffer and
loaded into the syringe automatically. Titration was carried out with 18 injections over a period of 40
min with stirring at 1000 rpm.
2.6 Protein relaxation analysis
The
15
N longitudinal T1, and transverse T2 relaxation rates and backbone 1H-15N-heteronuclear NOE
(hetNOE) experiments [26] were collected at 298 K using a purified pGyrB sample in the absence and
presence of the inhibitor at a Bruker Avance 700 MHz magnet. For T1 measurements, the relaxation
delays of 100, 300, 500, 1000, 1400, 1800, 2000, 2500 and 3000 ms were recorded. For T2
measurements, the data were acquired with delays of 16.9, 34, 51, 68, 85, 102, 119, 136 and 153 ms.
The hetNOE was obtained using two datasets that were collected with and without initial proton
saturation for a period of 3 s. The collected spectra were then processed with NMRPipe [22] and
analyzed with NMRView [23].
5
3. Results
3.1 NMR spectra of pGyrB
Structural studies of GyrB from E. coli revealed that the N-terminal ATP binding domain contains eight strands backed on the side with several helices [27]. Free pGyrB exhibited well dispersed cross peaks in
H-15N-TROSY spectrum (Fig. 1A), which also suggested that it constrains -strands. The OD280/OD260 of
1
purified protein was approximately 0.6, suggesting that pGyrB sample does not contain any nucleotides.
Protein aggregation was observed when the sample was kept at room temperature for more than two
days. Although 3D NMR experiment data were collected for backbone assignment, the data quality was
not good enough to complete the assignment using the conventional strategy due the weak signal in the
HNCACB experiment (Fig. 1B). In the presence of bis-pyridylurea, a potent inhibitor of both ParE and
GyrB of E. coli [21], chemical shift perturbation was observed, suggesting that pGyrB binds to the
inhibitor in solution (Fig. 1A). The protein-inhibitor complex was stable for more than seven days and
the quality of the 3D spectra was improved (Fig. 1B), which made the backbone assignment possible.
Both thermal shift assay (TSA) and ITC were carried out to understand protein-inhibitor interactions. TSA
showed that thermal shift (ΔTm) caused by inhibitor binding was more than 9 C (Fig. 2A). ITC result
suggested that the binding affinity (KD) was 54.6 nM and reaction stoichiometry (n) was approximately 1
(Fig. 2B), which explained the interaction was undergoing slow exchange observed in the NMR study (Fig.
1).
3.2 Backbone assignment of free pGyrB and complex
Backbone resonance assignment for the pGyrB-inhibitor complex was obtained using conventional 3D
experiments. The assignments of the 1H-15N-TROSY spectra of pGyrB in the absence and presence of the
inhibitor are shown in Figure 3A. Most of the backbone amides and amide protons were assigned except
M1, L100 and V120. Other backbone resonance assignments including Cα (218 of 222), C (190 of 198)
and C’ (217 of 222) have been obtained. The assignment of free pGyrB was achieved by referring to the
assignment of the complex. The assignments of free and inhibitor-bound pGyrB have been deposited in
the Biological Magnetic Resonance Bank (BMRB) with accession numbers 26597 and 26598, respectively.
Compared with the 1H-15N-TROSY spectrum of free pGyrB, no extra peaks appeared and no linebroadening was observed in the 1H-15N-TROSY spectrum of the pGyrB-inhibitor complex because the
interaction was undergoing slow exchange (Fig. 1A, Fig. 2).
6
3.3 Secondary structural analysis of pGyrB and the complex
The secondary structure analysis for pGyrB in the absence and presence of the inhibitor was conducted
using TALOS+ [24]. Both forms showed similar secondary structural elements to E. coli GyrB (Fig. 3C).
There are eight  strands including 1 (residues C58-I65), 2 (residues S70-N76), 3 (residues E131R138), 4 (residues K141-H148), 5 (residues L156-T162), 6 (residues S165-F171), 7 (residues V202D208) and 8 (residues K213-E220) and five α helices including α1 (residues L18-M27), α2 (residues T36A55) α3 (residues A92-T98), α4 (residues V122-L128) and α5 (residues W185-L198) present in the pGyrB.
The pGyrB shares very high sequence homology (more than 75% sequence identity) with GyrB of the E.
coli (eGyrB). The secondary structures of pGyrB derived from NMR data are similar to X-ray structure of
eGyrB, except that there is a short helix present at the N-terminus of eGyrB, and the lengths of α1, α2,
β1, α4, β3, β5, β6, and β8 are slightly different (Fig. 3C). There are several residues that are different
between these two proteins (Fig. 3C). Although the difference did not alter the structure of pGyrB, it
may affect inhibitor binding because some of different residues are at the inhibitor binding regions (Fig.
3C). A homology model of pGyrB was built using the SWISS-MODEL server (Fig. 3D) using the X-ray
structure of eGyrB bound with adenylyl-imidodiphosphate (ADPNP) as a template [2,28]. Long-range
NOEs of residues in the -strands of pGyrB were observed, which supports the homology model (data
not shown).
3.4 The bis-pyridylurea inhibitor binds to the ATP binding pocket
To determine which residues were affected by inhibitor binding to pGyrB, CSP caused by inhibitor
binding was plotted against residue number (Fig. 4A). As the chemical shifts of amide and amide protons
are sensitive to the environment, residues showing significant CSP might be involved in inhibitor binding.
It was clear that those residues from the α2, β2, β6, the loop between β2 and α3, α3 and α4 were
important for inhibitor interaction (Fig. 4A). Whether the inhibitor could cause structural changes on
pGyrB was investigated by analyzing the changes of the C chemical shifts that are sensitive to the
secondary structures. Although several residues showed changes in the C chemical shifts (Fig. 4B), the
overall structure of pGyrB was not altered as analyzed by TALOS+ (Fig. 3). The residues from 2
including 44 to 52 are affected significantly in the presence of the inhibitor, suggesting that they are
critical for inhibitor binding. This result may also explain the high quality HNCACB experiment obtained
for the complex because the inhibitor can affect the chemical environments of Cα and Cβ carbons.
Residues showing CSPs were mapped to the homology model of pGyrB (Fig. 4C). Compared with the X-
7
ray structure of the ParE-inhibitor complex, the inhibitor also binds to the ATP binding pocket of pGyrB
(Fig. 4D). NOEs between residues and inhibitor were also observed and the orientation of the inhibitor in
pGyrB is similar to the one in ParE of Streptococcus pneumonia (S. pn).
3.5 Backbone relaxation analysis of pGyrB and its complex
Backbone relaxation data T1, T2 and hetNOE revealed the dynamic properties of both free and inhibitor
bound pGyrB (Fig. 5). Compared with complex, less residues of free pGyrB were used for analysis
because of the peaks are too weak to be accurately analyzed. Surprisingly, there is no significant change
observed for these relaxation parameters in the absence and presence of the inhibitor. The N-terminal
25 residues and the C-terminal 5 residues are flexible in the absence and presence the inhibitor, which is
characterized with low T1 and hetNOE values and high T2 values (Fig. 5). The average T1 values excluding
both N- and C-terminal residues and the loop between α3 and α4 for free pGyrB and complex are 1.43 s
and 1.48 s, respectively. The average T2 values are 37.7 and 37.2 ms, respectively. The loop α3 and α4
compassing residues 100 to 120 is flexible for both free pGyrB and complex, suggesting that the loop is
not involved in the molecular interaction with this inhibitor. The hetNOE values of other residues are
higher than 0.82 that is expected for NH groups in a grid globular protein [26], indicating that these
residues are rigid in solution. Further dynamic study in other time scales or protein side chain relaxation
study will be helpful for understanding the effect of inhibitor on GyrB dynamics.
4. Discussion
Due to the bacterial resistance to antibiotics, there is a great need to develop novel antibacterial agents.
The rate-limiting steps in the antibacterial discovery process are twofold [29]. First, it is important to
select a target that is not prone to resistance development and second, it is important to increase
chemistry diversity to overcome the barriers to bacterial entry [29]. Bacterial type II topoisomerases
have been proven to be a good target for antibacterial development due to their high sequence
homology among the pathogenic bacteria and low homology with eukaryotes [5,7]. Structure-based
drug design has been an important tool in the development of these novel inhibitors such as tricyclic
GyrB/ParE inhibitors and azaindole class of antibacterial agents [7,10,30]. Understanding protein and
inhibitor interaction is important in drug development. In this study, we carried out NMR studies on the
pGyrB. It is interesting that the assignment for the free pGyrB was challenging due to the protein
stability and low signal sensitivity (Fig. 1B), which might be the reason that there is no structural
information available for both ParE and GyrB from P. ae, an extremely important Gram-negative strain
8
with high pathogenicity. In the presence of the inhibitor that binds to pGyrB with a KD of 54.6 nM, the
protein stability was improved (Fig. 2A) and we obtained backbone resonance assignment for both free
and inhibitor bound forms of pGyrB (Fig. 3). The secondary structural elements of pGyrB in solution
were determined based on the assignment (Fig. 3). Although the inhibitor binds to pGyrB with an affinity
in nanomolar range, there was no significant secondary structural change observed for pGyrB,
suggesting that the inhibitor did not cause any backbone conformational changes on pGyrB. Further
relaxation results also demonstrated that the backbone dynamic of pGyrB was not changed dramatically
(Fig. 5). It has been noted that our 3D spectra and relaxation data suggest that the side chain dynamics
of residues in the ATP binding pocket might be important for ligand binding. In the presence of the
inhibitor, the backbone amide dynamics of pGyrB were not affected significantly, while the side chain
dynamics was influenced (Fig 1B, Fig 2A). These results imply that careful protein and ligand interaction
need to be studied in drug development because the binding affinities between the inhibitor and
eGyrB/ParE might be different from pGyrB due to the difference of residues in the ATP binding pocket.
Our study on the ParE of S. pn showed that swapping a single residue with a corresponding residue in P.
ae in the ATP binding pocket can affected inhibitor binding affinity (Kang et al, unpublished data). Our
study also confirmed that the loop between 3 and 4 of pGyrB was not involved in inhibitor binding
because there were no CSP observed and dynamic changes were minor in the presence of the inhibitor
(Fig. 5).
The inhibitor used in this study is a pyridylurea scaffold derived from fragment-based drug design that is
ATP competitive [21]. Its activity against gram-negative strains such as E. coli and gram-positive strains
was investigated in detail [21]. X-ray crystal structure revealed that D78 of 2 and T172 of 6 from ParE
of S. pn formed hydrogen bonds with the inhibitor. R81 and M83 from S. pn ParE from the loop between
2 and α3 were shown to interact with the inhibitor [21]. We carried out biophysical characterization for
the molecular interaction between pGyrB and the inhibitor (Fig. 1 and Fig. 2). Based on the backbone
resonance assignment, the CSP caused by inhibitor binding was investigated in this study. Residues from
2 (D75), 6 (E168 and V169), loop between 2 and α3, and α3 were shown to be important for
inhibitor binding (Fig. 4). Residues exhibited CSP with more than 0.2 ppm upon inhibitor binding were
localized at the ATP binding pocket (Fig. 4). NOEs between pGyrB and the inhibitor were observed (Fig.
4), which further confirmed the residues that are important for inhibitor binding. Our results provide
direct evidence to show that the bis-pyridylurea inhibitor binds to the ATP binding pocket of pGyrB,
which is similar to the ParE of S. pn. These results will be useful to understand protein and inhibitor
9
interactions, which will be helpful in antibacterial drug development. Although it will be useful to carry
out further studies to understand protein dynamic in other time scales, this study provides an example
to show that inhibitors or ligands can facilitate structural studies of proteins by improving spectral
quality.
In summary, we purified pGyrB and conducted structural studies and its interaction with a bispyridylurea inhibitor. This inhibitor binds to pGyrB with a KD of 54.6 nM and could improve its thermal
stability. Secondary structures of pGyrB were defined and inhibitor did not cause significant
conformational changes. CSP and NOE analysis demonstrate that the inhibitor binds to the ATP binding
pocket.
Acknowledgments
We appreciate 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. The authors
appreciate valuable discussion from members of the drug discovery team in Experimental Therapeutics
Centre and AstraZeneca.
10
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12
Figure legends
Figure 1. NMR spectra of pGyrB. A. 1H-15N-TROSY spectra of pGyrB. The NMR spectra of pGyrB in the
absence (black) and presence (red) of the inhibitor were collected and superimposed, and two peaks
that undergo significant shifts upon complex formation are highlighted. Inside spectra are selected
regions of spectra with inhibitor/protein ratios of 0 (black), 0.5 (green) and 1 (red), respectively. The
interaction is undergoing slow exchange. B. 3D-HNCACB of pGyrB in the absence and presence of the
inhibitor. Select strips of HNCACB spectrum for several residues are shown. Upper and lower panels are
free and inhibitor-bound pGyrB, respectively. The peaks are labeled with residue number and atom
types.
Figure 2. TSA and ITC analysis. A. TSA of pGyrB in the absence and presence of the inhibitor. Effect of
DMSO, different concentrations of inhibitor on the thermal stability of pGyrB is plotted. B. ITC of the
inhibitor against pGyrB. The binding constant was 54.6 nM.
Figure 3. Assignments and secondary structural analysis for pGyrB and its complex. A, B. Assignment of
the 1H-15N-TROSY spectra of pGyrB in the absence (B) and presence of the inhibitor (A). Right panel is the
enlarged region of the box in the left panel. Residues specific assignment of backbone 1H and
15
N
frequencies is shown with residue name and sequence number. C. Secondary structure analysis of pGyrB.
Box indicates helical structures. Arrow indicates strands and line indicates loops. Secondary structural
elements for free pGyrB derived from NMR study and eGyrB derived from an X-ray structure (PDB ids
1EI1 and 4PRX) are shown in black and red, respectively. The inhibitor-bound pGyrB has the same
secondary structural elements as free pGyrB. The sequence alignment was conducted using ClustalW
(http://www.ebi.ac.uk/Tools/msa/clustalw2/). The different residues between eGyrB and pGyrB are
highlighted in red. D. Homology model of pGyrB. A model was built using structure of GyrB of E. coli as a
template. Left panel is the crystal structure of eGyrB (PDB id 1EI1). The ADPNP is show in sticks. Middle
panel is surface representation of the eGyrB-ADP complex. The right panel is the model of pGyrB.
Figure 4. Residues involved in protein and inhibitor interactions. A. Plot of CSP as a function of residue
number. B. Difference of C chemical shifts in the absence and presence of the inhibitor. The chemical
shift dereference ΔC was plotted against residue number. ΔC= ΔC(free)- ΔC(inhibitor). B. Mapping
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of affected residues on the pGyrB model. Residues with CSP more than 0.3 ppm, between 0.2 and 0.3
ppm and between 0.1 and 0.2 ppm are labeled in red, brown and yellow, respectively. The loop between
3 and 4 is shown in blue. D. Model of pGyrB and inhibitor complex. Left panel is the model of the
pGyrB and inhibitor complex, which was based on the structure of ParE-inhibitor complex (PDB id 4LP0).
Dashed lines indicate NOEs observed in the NOE experiment. The color code is similar to Fig. 4C.
Residues with unambiguous NOEs with the inhibitor are highlighted with boxes. Middle panel is the
structure of the inhibitor used in this study. Carbons with unambiguous assignments are labeled. Right
panel contains select strips of the NOESY-TROSY spectrum. The resonances that may arise from
incomplete deuteration or ambiguous assignments of the inhibitor are labeled with question marks. The
NOESY-TROSY spectrum was collected using a 13C/15N/2H-labeled pGyrB and inhibitor at a molar ratio of
1:2.
Figure 5 15N relaxation parameters for pGyrB in free (○) and inhibitor bound forms (▲). Unanalyzed
residues include prolines and the ones that are overlapped or too weak to be quantified. The relaxation
experiments were collected using a sample that contained 0.6 mM
absence and presence of 1.2 mM bis-pyridylurea inhibitor.
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C/15N/2H-labeled pGyrB in the
Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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