1H, 15 N & 13 C resonance Backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA

1H, 15 N & 13 C resonance Backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 1H, 15 N & 13 C resonance Backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA Jayanti Vaishnav, Ravi Sankar Ampapathi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4003535/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2024 Read the published version in Biomolecular NMR Assignments → Version 1 posted 6 You are reading this latest preprint version 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, reveals a well-folded structure and we present a 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. BRCT domain LigA Mycobacterium tuberculosis NAD+ DNA repair Figures Figure 1 Figure 2 Biological Context Tuberculosis remains a formidable global health challenge, claiming millions of lives annually, highlighting the enduring adaptability and survival capabilities of its causative agent, Mycobacterium tuberculosis ( Mtb ) (World Health Organization, 2023). The emergence of antibiotic resistance in Mtb strains, particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms, poses a serious threat, necessitating urgent exploration of novel therapeutic targets (Davies J and Davies D., 2010 ; Shah N. S. et al. 2007; Sotgiu G. et al. 2009 ). In Mtb , the NAD+-dependent DNA ligase is vital for maintaining genomic integrity by catalyzing the formation of phosphodiester bonds at single-strand breaks in double-stranded DNA (Wilkinson A. and Day J. 2001; Shuman S. and Lima C.D. 2004; Dwivedi N. et al. 2008 ). Structural and functional analyses of LigA reveal its highly modular nature, undergoing significant conformational changes during the catalytic cycle. Notably, the C-terminal BRCT domain approaches the N-terminal Adenylation domain, completing enzyme catalysis, underscoring the critical role of the C-terminal BRCT domain in this process (Lee J.Y. et al. 2000). This domain emerges as a potential novel target, with designed inhibitors capable of disrupting its interaction with the Adenylation domain, thereby affecting DNA replication and, consequently, the survival of Mtb (Miggiano R. et al. 2020 ; Gong C. et al. 2004 ; Dube D. et al. 2005; Srivastava S. K. et al. 2005). The BRCT domain, belonging to the BRCT domain superfamily, comprises a four-stranded parallel β -sheet flanked by three α -helices (Peña-Guerrero J. et al. 2023 ; Zhang X. et al. 1998 ; Callebaut I. et al. 1997). Engaging in constitutive interactions with other protein domains, the BRCT domain play a pivotal role in the recruitment of DNA ligase complexes to DNA damage sites through direct protein–protein interactions involving BRCT motifs. It is proposed that BRCT domains may function as signal transducers in DNA repair mechanisms through specific protein–protein interactions (Khanam T. et al. 2020 ). The crystal structure of Tfi ligase indicates the proximity of the BRCT domain to a conserved segment around the N-terminus of helix B in sub-domain 1a, potentially playing a role in forming a toroidal conformation (Lee J.Y. et al 2000). In the context of the NAD+-dependent ligase from Mtb , the absence of the BRCT domain significantly reduces enzyme activity, emphasizing its crucial role in the enzyme functionality (Srivastava S. K. et al. 2007). While well-established for protein-protein interactions, emerging evidence suggests the involvement of BRCT domains in DNA binding, particularly in bacterial NAD+-dependent DNA ligases (Fernandes A. et al. 2023 ). Deletion or mutation of the BRCT domain in these ligases results in diminished nick repairing and a substantial reduction in the transfer of the adenylate moiety to the 5'-terminal phosphate (Wang L K et al. 2008). The importance of inhibiting LigA activity during the initial stages of Base Excision Repair (BER), where XthA interacts with the BRCT domain, thus maintains extended conformation of LigA (Khanam T. et al. 2020 ). Determining the structure of the BRCT domain will enhance our comprehension of these pivotal domains, especially to dwell into the protein-protein interactions (PPI) and in exploring the inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. Mtb DNA ligase (LigA) is 691 amino acid long protein with BRCT spanning from 607 to 691 at the C-terminus of the protein. Attempts to solve the crystal structure of Mtb LigA revealed disorderness in the BRCT domain, and therefore the structure of BRCT domain of Mtb LigA is not available. Here, we present, for the first time, a nearly complete 1 H, 13 C, and 15 N resonance assignment of the BRCT domain of Mtb LigA. These assignments are expected to contribute valuable insights in exploring the role of BRCT in DNA repair in Mtb and serve as starting points for developing suitable inhibitors targeting the BRCT:Adenylation domain interactions, as well as in exploring other protein-protein interactions. Methods and Experiments Expression constructs The gene corresponding to the C-terminal BRCT domain (residues 605 to 691) of NAD + -dependent LigA of Mycobacterium tuberculosis ( Mtb ) was cloned in between EcoR I and Hind III restriction sites of Multiple cloning site of plasmid vector pET-23a+(Novagen). This gene construct was obtained as a kind gift from Dr. Ramachandran’s lab. Sequencing result has shown two mutations (Ser607Arg and Val667Ala) which were reverted back to the wild type sequence using site-directed mutagenesis (NEB-Q5 site directed mutagenesis kit) in two steps; reverting one mutation at a time using suitable primers. Set of primers for this two-step site directed mutagenesis were obtained from IDT and results for mutations were further confirmed by sequencing by IDT, Maptech. Expression and Purification of Isotopically labelled BRCT of Mtb LigA The BRCT domain of Mtb LigA was cloned into the pET23(a) plasmid and expressed in E. coli strain BL21(DE3) Rosetta pLysS. For the production of uniformly 15 N and 13 C labelled protein, cells were cultivated in standard M9 minimal medium having 15 NH4Cl (1 g/L) and 13 C-D-glucose (2 g/L) as the sole source of nitrogen and carbon, respectively. The media was supplemented with 100µg/mL ampicillin and 35 µg/ml chloramphenicol. Initially, a 10 mL overnight primary culture was grown at 37 °C with shaking at 180 rpm. This primary culture was used as inoculum to seed one litre culture, and cells were grown at 37°C, shaking at 180 rpm until the OD 600 reached 0.6. Protein expression was then induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and cells were further incubated at 16°C, shaking at 180 rpm for 16–18 hours. The cells were gently harvested by centrifugation at 3600 rpm, and the collected cell pellets were resuspended in Tris (50mM) buffer containing 200 mM NaCl, 0.5 mM PMSF protease inhibitor. Subsequently, 500 µL-1 mL bugbuster was added, and pellets were incubated at 4 °C for 20–30 minutes. Cells were then lysed using ultrasonication at 4°C for 15–20 minutes, employing a 10s on/off cycle. The lysed cell broth underwent high-speed centrifugation at 10000 rpm for 30–40 minutes at 4 ⁰C, separating the supernatant containing soluble proteins from the pellet of lysed cell debris. The overexpressed 15 N/ 13 C labelled BRCT protein was purified further using a standard two-step Chromatographic protocol involving Ni-NTA affinity chromatography and Size-exclusion chromatography. First step of purification was carried out by affinity method using Ni-NTA agarose and further purification was carried out by using the ÄKTA pure chromatography system (GE Healthcare) with a HiLoad Superdex S75 (16/60) column (GE Healthcare) in a 50 mM Tris at pH 6.5, 200 mM NaCl buffer. The purified protein was concentrated to 500 µL using 3KDa cut-off centrifugal devices and exchanged into an NMR buffer composed of 50 mM d -Tris at pH 6.5, 200 mM NaCl, 0.5 mM NaN 3 . The expressed recombinant protein contains 116 amino acids having molecular weight of 12.13 KDa. First 16 amino acid at N –terminal are from the plasmid, followed by 87 amino acids from D17 to T103 corresponding to D605 to T691 of BRCT domain of Mtb -LigA, 7 amino acid and 6X-HIS-tag at C-terminal as a part of the expression construct from plasmid. NMR spectroscopy All NMR experiments on BRCT protein were acquired on 700MHz NMR spectrometer, equipped with cryogenic triple resonance probe with 13 C enhancement using Vnmrj interface at 293K. The data processing was carried out using NmrPipe and nmrDraw (Delaglio et al. 1995 ). Data analysis was done using NMRFAM-SPARKY (Lee et al. 2014 ). Backbone chemical shift assignments followed by side-chain chemical shift assignments has been obtained by acquiring series of 2D and 3D-triple resonance NMR experiments like 2D 1 H- 15 N HSQC, 1 H- 13 C HSQC, 3D HNCACB, HNCO, HC(CO)NH-TOCSY, CC(CO)NH-TOCSY, HCCH-COSY, and CCH-TOCSY, [ 15 N, 1 H] TOCSY-HQSC, [ 15 N, 1 H]-NOESY-HSQC and [ 13 C, 1 H] NOESY-HSQC. Chemical shift referencing has been done using TSP- d4 (deuterated (3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid)) as an external reference. 1 H chemical shifts of water peak was referenced against TSP peak at 0 ppm, 13 C and 15 N Chemical shifts were referenced indirectly using 1 H chemical shift (Wishart et al. 1995 ). Extent of Assignment and data deposition C-terminal BRCT domain of LigA of Mtb consists of 87 amino acid containing 4 proline residues. For 83 non-proline residues, all 83 amide protons ( 1 HN), nitrogens ( 15 N) were assigned. For Carbon ( 13 C) out of 87 amino acid residues 82 C′(CO) i.e., 94.25% carbonyls could be assigned while C α and C β could be assigned for all of the 87 residues. For Side-chain assignment, more than 98% 13 C and 1 H resonances could be assigned, among them maximally all aliphatic side chains were assigned however for aromatic side chains –two Tyr and two Phe aromatic 13 C and 1 H resonances could not be unambiguously assigned. Side chain NH 2 of Asn could only be assigned. The chemical shift assignments ( 1 H, 15 N, 13 C) were deposited in BMRB (BioMagResBank– http://www.bmrb.wisc.edu/ ) under the accession number-52237. Chemical shift assignment: All the 2D, 3D-NMR Experimental data were processed and converted into the ucsf file format by using NMRPipe/Draw package. Data visualization, analysis and chemical shift assignments were carried out using NMRFAM sparky. 2D 1 H- 15 N HSQC, 3D HNCACB, HNCO were used to assign 1 H, 15 N and 13 C backbone chemical shifts, while side chain chemical shifts were assigned using 13 C-HSQC, CCONH, HCCONH, HSQCTOCSY with substantial help from HCCH-COSY and CCHTOCSY, 13 C-NOESY and 15 N-NOESY. The assigned 2D 1 H 15 N-HSQC spectrum is shown in Fig. 1. Side-chain resonances were assigned using 3D HC(CO)NH-TOCSY, CC(CO)NH-TOCSY, HCCH-TOCSY, and CCH-TOCSY spectra. [ 15 N, 1 H] TOCSY-HQSC, [ 15 N, 1 H]-NOESY-HSQC, 3D [ 15 N, 1 H] NOESY-HSQC and [ 13 C, 1 H] NOESY-HSQC experiments were also used for further confirmation of the assigned chemical shifts. Secondary Structure: The chemical shift assignments- 1 HN, 15 N, 1 Hα, 13 Cα, 13 Cβ and 13 C′ were used to predict secondary structure in BRCT using analysis by TALOS-N software from Bax group (Shen and Bax 2013 ). This provides the information about the secondary structure conformations in the BRCT domain. This information was plotted as SS (secondary structure) propensity as shown in Fig. 2(a), which show the presence of three α-helices and four β-sheets. The random coil index-order parameter (RCI-S 2 ) (Berjanskii and Wishart 2005 ) was also analysed by TALOS-N, which was ~ 0.7 or more for maximum residues except for C-terminal residues, as presented in the Fig. 2(b), which indicates rigidity of the BRCT structure. Conclusion The detailed solution NMR spectroscopic data acquisition and analysis on BRCT domain has provided us with important structural details. The well dispersed NHSQC suggested that protein was present in well folded stable structure. The chemical shift data analysis provides us insight into secondary structure elements present in BRCT domain in solution. This protein consists of 3α-helices and 4 β-sheets, the three-dimensional structure calculations are in progress, which supposedly may provide significant insights in designing and developing novel anti-tubercular drugs. Declarations 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 XXXX. Author contributions: 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. Funding: This research was funded by the Institutional research funding of CSIR-Central Drug Research Institute to R.S.A. Data availability The chemical shift data for BRCT domain of Mtb LigA is available in BMRB under accession number 52237. Conflict of interest: The authors declare no conflict of interest. References Berjanskii MV, Wishart DS (2005) A simple method to predict protein fexibility using secondary chemical shifts. J Am Chem Soc 127(43):14970–14971 Callebaut I, Mornon JP (1997) From BRCA1 to RAP1: A widespread BRCT module closely associated with DNA repair. FEBS Lett 400:25–30 Davies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74(3):417–433 Dwivedi N, Dube D, Pandey J, Singh B, Kukshal V, Ramachandran R, Rama PT (2008) NAD(+)-dependent DNA ligase: a novel target waiting for the right inhibitor. Med Res Rev 28:545–568 Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293 Fernandes A, Williamson A, Matias PM, Moe E (2023) Structure/function studies of the NAD+-dependent DNA ligase from the poly-extremophile Deinococcus radiodurans reveal importance of the BRCT domain for DNA binding. Extremophiles 27(3):26 Gong C, Martins A, Bongiorno P, Glickman M, Shuman S (2004) Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J Biol Chem 279:20594–20606 Khanam T, Afsar M, Shukla A, Alam F, Kumar S, Soyar H, Dolma K, Pasupuleti M, Srivastava KK, Ampapathi RS, Ramachandran R (2020) M. tuberculosis class II apurinic/ apyrimidinic-endonuclease/3'-5' exonuclease (XthA) engages with NAD+-dependent DNA ligase A (LigA) to counter futile cleavage and ligation cycles in base excision repair. Nucleic Acids Res 48(8):4325–4343 Lee J, Young C, Changsoo SH, Kyu M, Jinho Y, Jin KH-K Kwon Suk-Tae, Suh Se Won (2000) Crystal structure of NAD+-dependent DNA ligase: Modular architecture and functional implications. EMBO J 19: 1119–1129 Lee W, Tonelli M, Markley JL (2014) NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31:1325–1327 Miggiano R, Morrone C, Rossi F, Rizzi M (2020) Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair. Molecules 25(5):1205 Peña-Guerrero J, Fernández-Rubio C, García-Sosa AT, Nguewa PA (2023) BRCT Domains: Structure, Functions, and Implications in Disease-New Therapeutic Targets for Innovative Drug Discovery against Infections. Pharmaceutics 15(7):1839 Shah NS, Wright A, Bai GH, Barrera L, Boulahbal F, Martin-Casabona N, Drobniewski F, Gilpin C, Havelkova M, Lepe R, Lumb R, Metchock B, Portaels F, Rodrigues MF, Rusch-Gerdes S, Deun AV, Vincent V, Laserson K, Wells C, Cegielski JP (2007) Worldwide emergence of extensively drug-resistant tuberculosis. Emerg Infect Dis 13:380–387 Shen Y, Bax A (2013) Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artifcial neural networks. J Biomol NMR 56:227–241 Sotgiu G, Ferrara G, Matteelli A, Richardson MD, Centis R, Ruesch-Gerdes S, Toungoussova O, Zellweger JP, Spanevello A, Cirillo D, Lange C, Migliori GB (2009) Epidemiology and clinical management of XDR-TB: a systematic review by TBNET. Eur Respir J 33:871–881 Shuman S, Lima CD (2004) The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases. Curr Opin Struct Biol 14:757–764 Srivastava SK, Dube D, Tewari N, Dwivedi N, Tripathi RP, Ramachandran R (2005) Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I. Nucleic Acids Res 33:7090–7101 Srivastava SK, Tripathi RP, Ramachandran R (2005) NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors. J Biol Chem 280:30273–30281 Srivastava SK, Dube D, Kukshal V, Jha AK, Hajela K, Ramachandran R (2007) NAD+-dependent DNA ligase (Rv3014c) from Mycobacterium tuberculosis: novel structure-function relationship and identification of a specific inhibitor. Proteins 69:97–111 Wang LK, Nair PA, Shuman S (2008) Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase. J Biol Chem 283(34):23343–23352 Wilkinson A, Day J, Bowater R (2001) Bacterial DNA ligases. Mol Microbiol :1241–1248 Wishart DS et al (1995) H, 13 C and 15 N chemical shift referencing in biomolecular NMR. J Biomol NMR. https://doi.org/10.1007/BF0021177 World Health Organization (2020) Global tuberculosis report 2023 Zhang X, Moréra S, Bates PA, Whitehead PC, Coffer AI, Hainbucher K, Nash RA, Sternberg MJE, Lindahl T, Freemont PS (1998) Structure of an XRCC1 BRCT domain: A new protein-protein interaction module. EMBO J 17:6404–6411 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 30 Apr, 2024 Read the published version in Biomolecular NMR Assignments → Version 1 posted Reviews received at journal 04 Apr, 2024 Reviewers agreed at journal 15 Mar, 2024 Reviewers invited by journal 15 Mar, 2024 Editor assigned by journal 02 Mar, 2024 Submission checks completed at journal 02 Mar, 2024 First submitted to journal 01 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4003535","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275940384,"identity":"88589f7b-db15-487b-a46a-e732c4aaad13","order_by":0,"name":"Jayanti Vaishnav","email":"","orcid":"","institution":"Sophisticated Analytical Instrumentation Facility \u0026 Research (SAIF-R), CSIR-Central Drug Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Jayanti","middleName":"","lastName":"Vaishnav","suffix":""},{"id":275940385,"identity":"fd70053f-d89d-4731-b2e4-132aed6ed0c7","order_by":1,"name":"Ravi Sankar Ampapathi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIie3PPQrCMBTA8VcCdVGzdhC9QkoHF2mvklBIF3sCBwtCO7r2GB7A4UEHl1xChLr2AAVN/ViTujnkB4EM+fNeABznT6E+SwD+ugCQkUn0WzIQhU7GWVcqROjj7FjlV/TOmwQmMzQmC7Vl6JVpXqs7Q2ilKMjcPC8AydErSH4KpP4LNhzIlJkT2nK92D5j7+SR2JMgRQS/4Z9kmGhNbvpZeQlr1QJyTEVpX0wcOuh3K1pJ0nUYJ5QqczIg/ffG9fGt7x3HcRyrJ+FzQU3ThwWwAAAAAElFTkSuQmCC","orcid":"","institution":"Sophisticated Analytical Instrumentation Facility \u0026 Research (SAIF-R), CSIR-Central Drug Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Ravi","middleName":"Sankar","lastName":"Ampapathi","suffix":""}],"badges":[],"createdAt":"2024-03-01 14:44:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4003535/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4003535/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12104-024-10175-5","type":"published","date":"2024-04-30T19:58:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52074116,"identity":"b5dea3a1-d287-4bd9-8591-f1d72e0fc796","added_by":"auto","created_at":"2024-03-06 09:02:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":188822,"visible":true,"origin":"","legend":"\u003cp\u003eAssigned \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectrum of BRCT of \u003cem\u003eMtb\u003c/em\u003e LigA, acquired on a 700MHz NMR spectrometer at 293K. Resonances of NHs of amide bonds were annotated with amino acid-one letter code. Extra sequences from the plasmid are not shown in the figure.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4003535/v1/51e6d19ff01766b80a1efeb1.png"},{"id":52074115,"identity":"59681a06-9b5f-44f1-97f1-4218113d110d","added_by":"auto","created_at":"2024-03-06 09:02:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81397,"visible":true,"origin":"","legend":"\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Secondary structure propensity of residues of BRCT; (\u003cstrong\u003eb\u003c/strong\u003e) RCI-S\u003csup\u003e2 \u003c/sup\u003eorder parameter as predicted by TALOS-N software.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4003535/v1/63d8c84ce903ea7e723e6434.png"},{"id":56043068,"identity":"aa8ce0d4-08c4-45d7-88db-ba8bd5f10b82","added_by":"auto","created_at":"2024-05-07 20:10:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":634065,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4003535/v1/414153f3-72b0-4757-b826-6915fcf6d71c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"1H, 15 N \u0026 13 C resonance Backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA","fulltext":[{"header":"Biological Context","content":"\u003cp\u003eTuberculosis remains a formidable global health challenge, claiming millions of lives annually, highlighting the enduring adaptability and survival capabilities of its causative agent, Mycobacterium tuberculosis (\u003cem\u003eMtb\u003c/em\u003e) (World Health Organization, 2023). The emergence of antibiotic resistance in \u003cem\u003eMtb\u003c/em\u003e strains, particularly multidrug-resistant (MDR) and extensively drug-resistant (XDR) forms, poses a serious threat, necessitating urgent exploration of novel therapeutic targets (Davies J and Davies D., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Shah N. S. et al. 2007; Sotgiu G. et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In \u003cem\u003eMtb\u003c/em\u003e, the NAD+-dependent DNA ligase is vital for maintaining genomic integrity by catalyzing the formation of phosphodiester bonds at single-strand breaks in double-stranded DNA (Wilkinson A. and Day J. 2001; Shuman S. and Lima C.D. 2004; Dwivedi N. et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Structural and functional analyses of LigA reveal its highly modular nature, undergoing significant conformational changes during the catalytic cycle. Notably, the C-terminal BRCT domain approaches the N-terminal Adenylation domain, completing enzyme catalysis, underscoring the critical role of the C-terminal BRCT domain in this process (Lee J.Y. et al. 2000). This domain emerges as a potential novel target, with designed inhibitors capable of disrupting its interaction with the Adenylation domain, thereby affecting DNA replication and, consequently, the survival of \u003cem\u003eMtb\u003c/em\u003e (Miggiano R. et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gong C. et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Dube D. et al. 2005; Srivastava S. K. et al. 2005). The BRCT domain, belonging to the BRCT domain superfamily, comprises a four-stranded parallel \u003cem\u003eβ\u003c/em\u003e-sheet flanked by three \u003cem\u003eα\u003c/em\u003e-helices (Pe\u0026ntilde;a-Guerrero J. et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhang X. et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Callebaut I. et al. 1997). Engaging in constitutive interactions with other protein domains, the BRCT domain play a pivotal role in the recruitment of DNA ligase complexes to DNA damage sites through direct protein\u0026ndash;protein interactions involving BRCT motifs. It is proposed that BRCT domains may function as signal transducers in DNA repair mechanisms through specific protein\u0026ndash;protein interactions (Khanam T. et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The crystal structure of \u003cem\u003eTfi ligase\u003c/em\u003e indicates the proximity of the BRCT domain to a conserved segment around the N-terminus of helix B in sub-domain 1a, potentially playing a role in forming a toroidal conformation (Lee J.Y. et al 2000). In the context of the NAD+-dependent ligase from \u003cem\u003eMtb\u003c/em\u003e, the absence of the BRCT domain significantly reduces enzyme activity, emphasizing its crucial role in the enzyme functionality (Srivastava S. K. et al. 2007). While well-established for protein-protein interactions, emerging evidence suggests the involvement of BRCT domains in DNA binding, particularly in bacterial NAD+-dependent DNA ligases (Fernandes A. et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Deletion or mutation of the BRCT domain in these ligases results in diminished nick repairing and a substantial reduction in the transfer of the adenylate moiety to the 5'-terminal phosphate (Wang L K et al. 2008). The importance of inhibiting LigA activity during the initial stages of Base Excision Repair (BER), where XthA interacts with the BRCT domain, thus maintains extended conformation of LigA (Khanam T. et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDetermining the structure of the BRCT domain will enhance our comprehension of these pivotal domains, especially to dwell into the protein-protein interactions (PPI) and in exploring the inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. \u003cem\u003eMtb\u003c/em\u003e DNA ligase (LigA) is 691 amino acid long protein with BRCT spanning from 607 to 691 at the C-terminus of the protein. Attempts to solve the crystal structure of \u003cem\u003eMtb\u003c/em\u003e LigA revealed disorderness in the BRCT domain, and therefore the structure of BRCT domain of \u003cem\u003eMtb\u003c/em\u003e LigA is not available. Here, we present, for the first time, a nearly complete \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC, and \u003csup\u003e15\u003c/sup\u003eN resonance assignment of the BRCT domain of \u003cem\u003eMtb\u003c/em\u003e LigA. These assignments are expected to contribute valuable insights in exploring the role of BRCT in DNA repair in \u003cem\u003eMtb\u003c/em\u003e and serve as starting points for developing suitable inhibitors targeting the BRCT:Adenylation domain interactions, as well as in exploring other protein-protein interactions.\u003c/p\u003e"},{"header":"Methods and Experiments","content":"\u003cp\u003e \u003cb\u003eExpression constructs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe gene corresponding to the C-terminal BRCT domain (residues 605 to 691) of NAD\u003csup\u003e+\u003c/sup\u003e-dependent LigA of Mycobacterium tuberculosis (\u003cem\u003eMtb\u003c/em\u003e) was cloned in between EcoR I and Hind III restriction sites of Multiple cloning site of plasmid vector pET-23a+(Novagen). This gene construct was obtained as a kind gift from Dr. Ramachandran\u0026rsquo;s lab. Sequencing result has shown two mutations (Ser607Arg and Val667Ala) which were reverted back to the wild type sequence using site-directed mutagenesis (NEB-Q5 site directed mutagenesis kit) in two steps; reverting one mutation at a time using suitable primers. Set of primers for this two-step site directed mutagenesis were obtained from IDT and results for mutations were further confirmed by sequencing by IDT, Maptech.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression and Purification of Isotopically labelled BRCT of\u003c/b\u003e \u003cb\u003eMtb\u003c/b\u003e \u003cb\u003eLigA\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe BRCT domain of \u003cem\u003eMtb\u003c/em\u003e LigA was cloned into the pET23(a) plasmid and expressed in E. coli strain BL21(DE3) Rosetta pLysS. For the production of uniformly \u003csup\u003e15\u003c/sup\u003eN and \u003csup\u003e13\u003c/sup\u003eC labelled protein, cells were cultivated in standard M9 minimal medium having \u003csup\u003e15\u003c/sup\u003eNH4Cl (1 g/L) and \u003csup\u003e13\u003c/sup\u003eC-D-glucose (2 g/L) as the sole source of nitrogen and carbon, respectively. The media was supplemented with 100\u0026micro;g/mL ampicillin and 35 \u0026micro;g/ml chloramphenicol. Initially, a 10 mL overnight primary culture was grown at 37 \u0026deg;C with shaking at 180 rpm. This primary culture was used as inoculum to seed one litre culture, and cells were grown at 37\u0026deg;C, shaking at 180 rpm until the OD\u003csub\u003e600\u003c/sub\u003e reached 0.6. Protein expression was then induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and cells were further incubated at 16\u0026deg;C, shaking at 180 rpm for 16\u0026ndash;18 hours. The cells were gently harvested by centrifugation at 3600 rpm, and the collected cell pellets were resuspended in Tris (50mM) buffer containing 200 mM NaCl, 0.5 mM PMSF protease inhibitor. Subsequently, 500 \u0026micro;L-1 mL bugbuster was added, and pellets were incubated at 4 \u0026deg;C for 20\u0026ndash;30 minutes. Cells were then lysed using ultrasonication at 4\u0026deg;C for 15\u0026ndash;20 minutes, employing a 10s on/off cycle. The lysed cell broth underwent high-speed centrifugation at 10000 rpm for 30\u0026ndash;40 minutes at 4 ⁰C, separating the supernatant containing soluble proteins from the pellet of lysed cell debris. The overexpressed \u003csup\u003e15\u003c/sup\u003eN/\u003csup\u003e13\u003c/sup\u003eC labelled BRCT protein was purified further using a standard two-step Chromatographic protocol involving Ni-NTA affinity chromatography and Size-exclusion chromatography. First step of purification was carried out by affinity method using Ni-NTA agarose and further purification was carried out by using the \u0026Auml;KTA pure chromatography system (GE Healthcare) with a HiLoad Superdex S75 (16/60) column (GE Healthcare) in a 50 mM Tris at pH 6.5, 200 mM NaCl buffer. The purified protein was concentrated to 500 \u0026micro;L using 3KDa cut-off centrifugal devices and exchanged into an NMR buffer composed of 50 mM \u003cem\u003ed\u003c/em\u003e-Tris at pH 6.5, 200 mM NaCl, 0.5 mM NaN\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe expressed recombinant protein contains 116 amino acids having molecular weight of 12.13 KDa. First 16 amino acid at N \u0026ndash;terminal are from the plasmid, followed by 87 amino acids from D17 to T103 corresponding to D605 to T691 of BRCT domain of \u003cem\u003eMtb\u003c/em\u003e-LigA, 7 amino acid and 6X-HIS-tag at C-terminal as a part of the expression construct from plasmid.\u003c/p\u003e\n\u003ch3\u003eNMR spectroscopy\u003c/h3\u003e\n\u003cp\u003eAll NMR experiments on BRCT protein were acquired on 700MHz NMR spectrometer, equipped with cryogenic triple resonance probe with \u003csup\u003e13\u003c/sup\u003eC enhancement using Vnmrj interface at 293K. The data processing was carried out using NmrPipe and nmrDraw (Delaglio et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Data analysis was done using NMRFAM-SPARKY (Lee et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Backbone chemical shift assignments followed by side-chain chemical shift assignments has been obtained by acquiring series of 2D and 3D-triple resonance NMR experiments like 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC, \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC, 3D HNCACB, HNCO, HC(CO)NH-TOCSY, CC(CO)NH-TOCSY, HCCH-COSY, and CCH-TOCSY, [\u003csup\u003e15\u003c/sup\u003eN,\u003csup\u003e1\u003c/sup\u003eH] TOCSY-HQSC, [\u003csup\u003e15\u003c/sup\u003eN,\u003csup\u003e1\u003c/sup\u003eH]-NOESY-HSQC and [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e1\u003c/sup\u003eH] NOESY-HSQC. Chemical shift referencing has been done using TSP-\u003csub\u003ed4\u003c/sub\u003e (deuterated (3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid)) as an external reference. \u003csup\u003e1\u003c/sup\u003eH chemical shifts of water peak was referenced against TSP peak at 0 ppm, \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e15\u003c/sup\u003eN Chemical shifts were referenced indirectly using \u003csup\u003e1\u003c/sup\u003eH chemical shift (Wishart et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eExtent of Assignment and data deposition\u003c/h3\u003e\n\u003cp\u003eC-terminal BRCT domain of LigA of \u003cem\u003eMtb\u003c/em\u003e consists of 87 amino acid containing 4 proline residues. For 83 non-proline residues, all 83 amide protons (\u003csup\u003e1\u003c/sup\u003eHN), nitrogens (\u003csup\u003e15\u003c/sup\u003eN) were assigned. For Carbon (\u003csup\u003e13\u003c/sup\u003eC) out of 87 amino acid residues 82 C\u0026prime;(CO) i.e., 94.25% carbonyls could be assigned while C\u003csub\u003eα\u003c/sub\u003e and C\u003csub\u003eβ\u003c/sub\u003e could be assigned for all of the 87 residues. For Side-chain assignment, more than 98% \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e1\u003c/sup\u003eH resonances could be assigned, among them maximally all aliphatic side chains were assigned however for aromatic side chains \u0026ndash;two Tyr and two Phe aromatic \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e1\u003c/sup\u003eH resonances could not be unambiguously assigned. Side chain NH\u003csub\u003e2\u003c/sub\u003e of Asn could only be assigned. The chemical shift assignments (\u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e15\u003c/sup\u003eN, \u003csup\u003e13\u003c/sup\u003eC) were deposited in BMRB (BioMagResBank\u0026ndash;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bmrb.wisc.edu/\u003c/span\u003e\u003cspan address=\"http://www.bmrb.wisc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) under the accession number-52237.\u003c/p\u003e\n\u003ch3\u003eChemical shift assignment:\u003c/h3\u003e\n\u003cp\u003eAll the 2D, 3D-NMR Experimental data were processed and converted into the ucsf file format by using NMRPipe/Draw package. Data visualization, analysis and chemical shift assignments were carried out using NMRFAM sparky. 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC, 3D HNCACB, HNCO were used to assign \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e15\u003c/sup\u003eN and \u003csup\u003e13\u003c/sup\u003eC backbone chemical shifts, while side chain chemical shifts were assigned using \u003csup\u003e13\u003c/sup\u003eC-HSQC, CCONH, HCCONH, HSQCTOCSY with substantial help from HCCH-COSY and CCHTOCSY, \u003csup\u003e13\u003c/sup\u003eC-NOESY and \u003csup\u003e15\u003c/sup\u003eN-NOESY. The assigned 2D \u003csup\u003e1\u003c/sup\u003eH \u003csup\u003e15\u003c/sup\u003eN-HSQC spectrum is shown in Fig.\u0026nbsp;1. Side-chain resonances were assigned using 3D HC(CO)NH-TOCSY, CC(CO)NH-TOCSY, HCCH-TOCSY, and CCH-TOCSY spectra. [\u003csup\u003e15\u003c/sup\u003eN,\u003csup\u003e1\u003c/sup\u003eH] TOCSY-HQSC, [\u003csup\u003e15\u003c/sup\u003eN,\u003csup\u003e1\u003c/sup\u003eH]-NOESY-HSQC, 3D [\u003csup\u003e15\u003c/sup\u003eN,\u003csup\u003e1\u003c/sup\u003eH] NOESY-HSQC and [\u003csup\u003e13\u003c/sup\u003eC,\u003csup\u003e1\u003c/sup\u003eH] NOESY-HSQC experiments were also used for further confirmation of the assigned chemical shifts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSecondary Structure:\u003c/h3\u003e\n\u003cp\u003eThe chemical shift assignments- \u003csup\u003e1\u003c/sup\u003eHN, \u003csup\u003e15\u003c/sup\u003eN, \u003csup\u003e1\u003c/sup\u003eHα, \u003csup\u003e13\u003c/sup\u003eCα, \u003csup\u003e13\u003c/sup\u003eCβ and \u003csup\u003e13\u003c/sup\u003eC\u0026prime; were used to predict secondary structure in BRCT using analysis by TALOS-N software from Bax group (Shen and Bax \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This provides the information about the secondary structure conformations in the BRCT domain. This information was plotted as SS (secondary structure) propensity as shown in Fig.\u0026nbsp;2(a), which show the presence of three α-helices and four β-sheets. The random coil index-order parameter (RCI-S\u003csup\u003e2\u003c/sup\u003e) (Berjanskii and Wishart \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) was also analysed by TALOS-N, which was ~\u0026thinsp;0.7 or more for maximum residues except for C-terminal residues, as presented in the Fig.\u0026nbsp;2(b), which indicates rigidity of the BRCT structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe detailed solution NMR spectroscopic data acquisition and analysis on BRCT domain has provided us with important structural details. The well dispersed NHSQC suggested that protein was present in well folded stable structure. The chemical shift data analysis provides us insight into secondary structure elements present in BRCT domain in solution. This protein consists of 3α-helices and 4 β-sheets, the three-dimensional structure calculations are in progress, which supposedly may provide significant insights in designing and developing novel anti-tubercular drugs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eJV 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 XXXX.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003eJV 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was funded by the Institutional research funding of CSIR-Central Drug Research Institute to R.S.A. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chemical shift data for BRCT domain of \u003cem\u003eMtb\u003c/em\u003e LigA is available in BMRB under accession number 52237.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBerjanskii MV, Wishart DS (2005) A simple method to predict protein fexibility using secondary chemical shifts. J Am Chem Soc 127(43):14970\u0026ndash;14971\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCallebaut I, Mornon JP (1997) From BRCA1 to RAP1: A widespread BRCT module closely associated with DNA repair. FEBS Lett 400:25\u0026ndash;30\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74(3):417\u0026ndash;433\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDwivedi N, Dube D, Pandey J, Singh B, Kukshal V, Ramachandran R, Rama PT (2008) NAD(+)-dependent DNA ligase: a novel target waiting for the right inhibitor. Med Res Rev 28:545\u0026ndash;568\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277\u0026ndash;293\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandes A, Williamson A, Matias PM, Moe E (2023) Structure/function studies of the NAD+-dependent DNA ligase from the poly-extremophile Deinococcus radiodurans reveal importance of the BRCT domain for DNA binding. Extremophiles 27(3):26\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong C, Martins A, Bongiorno P, Glickman M, Shuman S (2004) Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J Biol Chem 279:20594\u0026ndash;20606\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhanam T, Afsar M, Shukla A, Alam F, Kumar S, Soyar H, Dolma K, Pasupuleti M, Srivastava KK, Ampapathi RS, Ramachandran R (2020) M. tuberculosis class II apurinic/ apyrimidinic-endonuclease/3'-5' exonuclease (XthA) engages with NAD+-dependent DNA ligase A (LigA) to counter futile cleavage and ligation cycles in base excision repair. Nucleic Acids Res 48(8):4325\u0026ndash;4343\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee J, Young C, Changsoo SH, Kyu M, Jinho Y, Jin KH-K Kwon Suk-Tae, Suh Se Won (2000) Crystal structure of NAD+-dependent DNA ligase: Modular architecture and functional implications. EMBO J 19: 1119\u0026ndash;1129\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee W, Tonelli M, Markley JL (2014) NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics 31:1325\u0026ndash;1327\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiggiano R, Morrone C, Rossi F, Rizzi M (2020) Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair. Molecules 25(5):1205\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePe\u0026ntilde;a-Guerrero J, Fern\u0026aacute;ndez-Rubio C, Garc\u0026iacute;a-Sosa AT, Nguewa PA (2023) BRCT Domains: Structure, Functions, and Implications in Disease-New Therapeutic Targets for Innovative Drug Discovery against Infections. Pharmaceutics 15(7):1839\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShah NS, Wright A, Bai GH, Barrera L, Boulahbal F, Martin-Casabona N, Drobniewski F, Gilpin C, Havelkova M, Lepe R, Lumb R, Metchock B, Portaels F, Rodrigues MF, Rusch-Gerdes S, Deun AV, Vincent V, Laserson K, Wells C, Cegielski JP (2007) Worldwide emergence of extensively drug-resistant tuberculosis. Emerg Infect Dis 13:380\u0026ndash;387\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen Y, Bax A (2013) Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artifcial neural networks. J Biomol NMR 56:227\u0026ndash;241\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSotgiu G, Ferrara G, Matteelli A, Richardson MD, Centis R, Ruesch-Gerdes S, Toungoussova O, Zellweger JP, Spanevello A, Cirillo D, Lange C, Migliori GB (2009) Epidemiology and clinical management of XDR-TB: a systematic review by TBNET. Eur Respir J 33:871\u0026ndash;881\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShuman S, Lima CD (2004) The polynucleotide ligase and RNA capping enzyme superfamily of covalent nucleotidyltransferases. Curr Opin Struct Biol 14:757\u0026ndash;764\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrivastava SK, Dube D, Tewari N, Dwivedi N, Tripathi RP, Ramachandran R (2005) Mycobacterium tuberculosis NAD+-dependent DNA ligase is selectively inhibited by glycosylamines compared with human DNA ligase I. Nucleic Acids Res 33:7090\u0026ndash;7101\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrivastava SK, Tripathi RP, Ramachandran R (2005) NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors. J Biol Chem 280:30273\u0026ndash;30281\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrivastava SK, Dube D, Kukshal V, Jha AK, Hajela K, Ramachandran R (2007) NAD+-dependent DNA ligase (Rv3014c) from Mycobacterium tuberculosis: novel structure-function relationship and identification of a specific inhibitor. Proteins 69:97\u0026ndash;111\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang LK, Nair PA, Shuman S (2008) Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase. J Biol Chem 283(34):23343\u0026ndash;23352\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilkinson A, Day J, Bowater R (2001) Bacterial DNA ligases. Mol Microbiol :1241\u0026ndash;1248\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWishart DS et al (1995) H, 13 C and 15 N chemical shift referencing in biomolecular NMR. J Biomol NMR. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF0021177\u003c/span\u003e\u003cspan address=\"10.1007/BF0021177\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization (2020) Global tuberculosis report 2023\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Mor\u0026eacute;ra S, Bates PA, Whitehead PC, Coffer AI, Hainbucher K, Nash RA, Sternberg MJE, Lindahl T, Freemont PS (1998) Structure of an XRCC1 BRCT domain: A new protein-protein interaction module. EMBO J 17:6404\u0026ndash;6411\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biomolecular-nmr-assignments","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnmr","sideBox":"Learn more about [Biomolecular NMR Assignments](http://link.springer.com/journal/12104)","snPcode":"12104","submissionUrl":"https://submission.nature.com/new-submission/12104/3","title":"Biomolecular NMR Assignments","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"BRCT domain, LigA, Mycobacterium tuberculosis, NAD+, DNA repair","lastPublishedDoi":"10.21203/rs.3.rs-4003535/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4003535/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e (\u003cem\u003eMtb\u003c/em\u003e), 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 \u003cem\u003eMtb\u003c/em\u003e. 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 \u003cem\u003eMtb\u003c/em\u003e 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 \u003cem\u003eMtb\u003c/em\u003e LigA and conduct its analysis using solution NMR spectroscopy, reveals a well-folded structure and we present a nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 \u003cem\u003eα\u003c/em\u003e-helices and 4 \u003cem\u003eβ\u003c/em\u003e-sheets, closely resembling the typical structural topology of most BRCT domains.\u003c/p\u003e","manuscriptTitle":"1H, 15 N \u0026amp; 13 C resonance Backbone and side-chain assignments and secondary structure determination of the BRCT domain of Mtb LigA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-06 09:02:53","doi":"10.21203/rs.3.rs-4003535/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-04-04T10:28:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"05ff4072-5b75-454a-ad89-456b80152c61","date":"2024-03-16T02:27:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-16T02:18:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-02T07:22:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-02T07:22:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biomolecular NMR Assignments","date":"2024-03-01T14:20:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biomolecular-nmr-assignments","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnmr","sideBox":"Learn more about [Biomolecular NMR Assignments](http://link.springer.com/journal/12104)","snPcode":"12104","submissionUrl":"https://submission.nature.com/new-submission/12104/3","title":"Biomolecular NMR Assignments","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9b6060e5-736a-47c4-b896-00780359e03f","owner":[],"postedDate":"March 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-07T20:06:57+00:00","versionOfRecord":{"articleIdentity":"rs-4003535","link":"https://doi.org/10.1007/s12104-024-10175-5","journal":{"identity":"biomolecular-nmr-assignments","isVorOnly":false,"title":"Biomolecular NMR Assignments"},"publishedOn":"2024-04-30 19:58:19","publishedOnDateReadable":"April 30th, 2024"},"versionCreatedAt":"2024-03-06 09:02:53","video":"","vorDoi":"10.1007/s12104-024-10175-5","vorDoiUrl":"https://doi.org/10.1007/s12104-024-10175-5","workflowStages":[]},"version":"v1","identity":"rs-4003535","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4003535","identity":"rs-4003535","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}