Abstract
The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein–protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein–protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 β-sheets, closely resembling the typical structural topology of most BRCT domains.
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Data availability
The chemical shift data for BRCT domain of Mtb LigA is available in BMRB under accession number 52237.
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Acknowledgements
JV is thankful to ICMR, New Delhi for fellowship. Authors are thankful to DST for 700 MHz Facility of SAIF at CDRI. The CDRI communication number allotted to this manuscript is 10775.
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This research was funded by the Institutional research funding of CSIR-Central Drug Research Institute to R.S.A.
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JV and RSA designed the experiments; JV prepared the samples; JV and RSA collected the NMR experiments; JV analysed the experiments; JV and RSA discussed the results; JV wrote the manuscript, RSA edited and approved the final version.
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Vaishnav, J., Ampapathi, R.S. 1H, 15N and 13C resonance backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA. Biomol NMR Assign 18, 105–109 (2024). https://doi.org/10.1007/s12104-024-10175-5
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DOI: https://doi.org/10.1007/s12104-024-10175-5