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 15N and 13C labelled protein, cells were cultivated in standard M9 minimal medium having 15NH4Cl (1 g/L) and 13C-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 OD600 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 15N/13C 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 NaN3.
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 13C 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 1H-15N HSQC, 1H-13C HSQC, 3D HNCACB, HNCO, HC(CO)NH-TOCSY, CC(CO)NH-TOCSY, HCCH-COSY, and CCH-TOCSY, [15N,1H] TOCSY-HQSC, [15N,1H]-NOESY-HSQC and [13C,1H] NOESY-HSQC. Chemical shift referencing has been done using TSP-d4 (deuterated (3-(trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid)) as an external reference. 1H chemical shifts of water peak was referenced against TSP peak at 0 ppm, 13C and 15N Chemical shifts were referenced indirectly using 1H 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 (1HN), nitrogens (15N) were assigned. For Carbon (13C) out of 87 amino acid residues 82 C′(CO) i.e., 94.25% carbonyls could be assigned while Cα and Cβ could be assigned for all of the 87 residues. For Side-chain assignment, more than 98% 13C and 1H resonances could be assigned, among them maximally all aliphatic side chains were assigned however for aromatic side chains –two Tyr and two Phe aromatic 13C and 1H resonances could not be unambiguously assigned. Side chain NH2 of Asn could only be assigned. The chemical shift assignments (1H, 15N, 13C) 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 1H-15N HSQC, 3D HNCACB, HNCO were used to assign 1H, 15N and 13C backbone chemical shifts, while side chain chemical shifts were assigned using 13C-HSQC, CCONH, HCCONH, HSQCTOCSY with substantial help from HCCH-COSY and CCHTOCSY, 13C-NOESY and 15N-NOESY. The assigned 2D 1H 15N-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. [15N,1H] TOCSY-HQSC, [15N,1H]-NOESY-HSQC, 3D [15N,1H] NOESY-HSQC and [13C,1H] NOESY-HSQC experiments were also used for further confirmation of the assigned chemical shifts.
Secondary Structure:
The chemical shift assignments- 1HN, 15N, 1Hα, 13Cα, 13Cβ and 13C′ 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-S2) (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.