Structural insights into the transient closed conformation and pH dependent ATPase activity of S.Typhi GyraseB N- terminal domain

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Highlights

  • GyrB N-terminal domain (GBNTD), hydrolyzes ATP during the catalytic activity of DNA Gyrase

  • The three-dimensional structure of GBNTD from Salmonella Typhi adopts transient closed conformation at pH 8.0.

  • Polar interactions observed at dimeric interface and ATP binding site stabilise the overall protein.

  • Role of ionization states of polar amino acids investigated at different pH milieu by kinetics and spectroscopic assays.

Abstract

DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 Å resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.

Introduction

DNA Gyrase is a type II DNA topoisomerase family protein that regulates the topology transition of DNA by introducing negative supercoils into closed circular DNA [1]. The basic supercoiling reaction of DNA Gyrase involves the breaking, passing, and resealing of the DNA backbone, resulting in a change in the linking number of DNA. The two subunits each of GyrA and GyrB associate together to form a functional A2B2 tetramer [[2], [3], [4]]. GyrA subunit forms the cutting blades termed as the breaking resealing component while the GyrB subunit constitutes the ATP driven energy transduction component. GyrA subunit comprises the N-terminal domain (GANTD) which takes part in DNA breakage and reunion reaction [5] and the C-terminal domain (GACTD) that is involved in wrapping DNA [6]. GyrB subunit also consists of two domains, the GyrB N-terminal domain (GBNTD) subdivided into ATPase and transducer subdomains which is responsible for hydrolyzing ATP [7], and the C-terminal domain (GBCTD) that is involved in nucleotide binding [8].

The structure of GBNTD from E.coli has been determined in the presence of nucleotide and its analogues; AMP-PNP (PDB Id: 1EI1) [9], ADP.Pi (PDB Id: 4PRX) [10] and ADP.Mg2+ (PDB Id: 4PRV) [10] in different conformations. These structures indicate that upon binding with nucleotide, the two GBNTD subunits self-associate, forming an ATP actuated N gate that captures the substrate molecule in its binding cavity and attains a closed, open and semi-open conformation in the presence of AMP-PNP, ADP.Pi and ADP.Mg2+ respectively [11,12]. The differences observed in the three conformations of GBNTD are associated with the extension of a segment of transducer subdomain termed as switch loop (QTK loop) into the ATP binding site that positions itself into the proximity of γ-phosphate of ATP [13]. Interestingly, the crystal structures of E.coli GBNTD have revealed that the switch loop of transducer subdomain resides outside the binding cavity in absence of ATP. However, in the presence of ATP, the transducer subdomain rotates several degrees with respect to ATPase subdomain to insert the QTK loop near the ATP binding site [7]. These induced conformational changes act as a relay signal for the passage of the Transfer or T segment DNA duplex through a transient break in the Gate segment or the G segment of the DNA duplex [14].

The ATP hydrolysis plays an essential role in providing energy to GBNTD for carrying out gyrase mediated negative supercoiling reaction. This activation of ATP hydrolysis is achieved by the dimerization of GBNTD that is required for catalytic DNA transfer during the enzymatic process [15]. The coupling between ATP hydrolysis and DNA transport has also been investigated in various species including E. coli [9], Bacillus subtilis [16], Micrococcus luteus [17], Mycobacterial species [18], Drosophila melanogaster [19] and yeast topoisomerase II [4]. DNA Gyrase is a very attractive drug target for the development of antibacterial drugs, that is essential for bacterial cell survival and absent in higher eukaryotes. The significance of GBNTD as an efficient drug target has led to several studies from different species related to its structural, functional and mechanistic behaviour. As DNA gyrase is a highly dynamic protein, analysis of this new transient conformation will aid to provide further insights about the molecular mechanism involving various steps of ATP hydrolysis. This study also illustrates the effect of different pH environment on the secondary and tertiary structure as well as its ATPase activity. The change in pH milieu results in different ionization states of the side chains of polar amino acid residues which in turn leads to loss of secondary and tertiary structure and ultimately loss of activity of the protein. Though earlier pH based studies for GyrB E.coli, Bacillus subtilis and M.smegmatis indicate that the protein has optimum activity between pH values 7.0–8.5 [20,21], the complete kinetic analysis of ATPase activity from pH 2.0–11.0 has not been reported so far. Here we report, for the first time the crystal structure of transient state conformation of GBNTD from Salmonella Typhi (StGBNTD) in complex with ADP and a Mg2+ ion at 1.94 Å resolution. We also characterized the ATPase activity of StGBNTD at broad range pH conditions (2–11) and correlated with protein's secondary and tertiary structure monitored by far UV CD (circular dichroism) and fluorescence spectroscopy. Since, DNA Gyr B subunit has been comprehensively exploited as one of the drug targets [22], knowledge regarding its structure, stability and mechanism of action at different pH environment will help to target this protein to develop novel druggable anti-infective agent against bacteria. The present study elucidates the conformational behavior of GBNTD from S.Typhi in the presence of ATP which is crucial for its activity.

Section snippets

Materials and methods

Luria broth and agar media were obtained from Difco (BD Biosciences). Superdex S-200 pre-packed columns were purchased from Cytiva, Uppsala, Sweden. Millipore syringe filters (0.22 μm) were procured from Millipore Corporation (USA). Pi ColorLockGold phosphate detection kit was purchased from Innova Biosciences (Cambridge, UK). All other chemicals used in the experiments are of molecular grade and were obtained from Sigma Chemical Co. (St. Louis, USA).

Purification and characterization of recombinant His-tagged StGBNTD protein

Recombinant StGBNTD was purified using Ni-NTA metal ion chromatography followed by gel filtration chromatography, suggesting that the protein exists as a monomer (Fig. 1a). The protein was further subjected to thermal experiments using both far-UV CD and DSC in a buffer containing 25 mM Tris pH 8.0 and 150 mM NaCl (Fig. 1bc). The thermodynamic parameters of StGBNTD obtained from CD were Tm=44.2 °C, ΔΗm=89.4 kcal mol−1, ΔCp = 1.9 kcal mol−1K−1and ΔGD0 = 4.30 kcal mol−1 while the calorimetric

Discussion

DNA Gyrase is an efficient drug target that catalyses negative supercoiling of double-stranded DNA by utilizing energy generated by ATP hydrolysis. GBNTD consists of ATPase and transducer subdomains that are essential for ATP hydrolysis as well as for DNA capture during the enzymatic process. Binding of ATP molecule induces conformational changes that are necessary for the conversion of the open clamp-like structure into a closed clamp protein conformation that can capture DNA molecule and

Conclusion

A high degree of conservation in the ATP binding site makes gyrase an appealing target for inhibitor development as it is less prone to resistance mutations. The GyrB subunit undergoes conformational changes during ATP hydrolysis. Based on the degree of rotation of transducer subdomain over ATPase subdomain and the inter-subunit distances measured between the two monomers, our present work reveals a new (transient closed) dimeric conformation of StGBNTD that lies between the two observed closed

Credit authorship contribution statement

DG: Contributed to experimental conception, design and data analysis, manuscript writing, reviewing & editing; PT: Conceptualizing and performing crystallization experiment, structure determination and refinement, manuscript writing, reviewing & editing; MAH: Contributed to fluorescence and CD data collection analysis and curation; ES: helped with experimental work and crystal data collection; MIH: Providing instrumentation facility for DSC data collection and design of experiment; ASE:

Ethical approval

This article does not involve any study with human participants or animals to be performed by any of the authors.

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgements (Funding)

Grant of funding from Indian Council of Medical Research, New Delhi is gratefully acknowledged. DG and PT thank AIIMS, New Delhi and ICMR respectively for grant of fellowship.

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