Structural and mutational analysis of MazE6-operator DNA complex provide insights into autoregulation of toxin-antitoxin systems

Abstract

Of the 10 paralogs of MazEF Toxin-Antitoxin system in Mycobacterium tuberculosis, MazEF6 plays an important role in multidrug tolerance, virulence, stress adaptation and Non Replicative Persistant (NRP) state establishment. The solution structures of the DNA binding domain of MazE6 and of its complex with the cognate operator DNA show that transcriptional regulation occurs by binding of MazE6 to an 18 bp operator sequence bearing the TANNNT motif (-10 region). Kinetics and thermodynamics of association, as determined by NMR and ITC, indicate that the nMazE6-DNA complex is of high affinity. Residues in N-terminal region of MazE6 that are key for its homodimerization, DNA binding specificity, and the base pairs in the operator DNA essential for the protein-DNA interaction, have been identified. It provides a basis for design of chemotherapeutic agents that will act via disruption of TA autoregulation, leading to cell death.

The dimeric MazE6 antitoxin binds to a specific sequence in its cognate operator DNA for autoregulation, and the key residues for dimerization and DNA binding are identified.

Introduction

Toxin-Antitoxin (TA) systems are stress response systems found on plasmids and chromosomes of bacteria and archaea^1–3. They are involved in important functions such as plasmid maintenance^2,4, programmed cell death^5,6, persister cell formation^7–9, phage abortive infection¹⁰, bacteriostasis and formation of antibiotic-resistant population^11,12. Based on the mode of action and chemical nature of the antitoxin, TA systems are divided into eight classes, Type I–Type VIII¹³. Among these, Type II system is the most widely represented and also the most extensively characterized^14,15. According to TADB2.0 database generated in 2017, there are 105 experimentally validated Type II TA pairs¹⁶.

The TA systems in Mycobacterium tuberculosis (Mtb) are often attributed to growth, persister cell formation and pathogenicity^14,17. Under conditions of stress the H37Rv strain of Mtb persists in host granulomas in a non-replicating and drug-tolerant state for extended periods of time and causes disease when the stress is relieved. Several free-living bacteria such as Mtb and Nitrosomonas europaea are known to encode for a large number of TA systems¹. The H37Rv strain of Mtb encodes an exceptionally high number (≈90) of TA systems, while the non-pathogenic mycobacterial species, M. smegmatis encodes only 5 TA systems and is relatively fast growing^18,19. The absence of TA loci in humans and their specific presence in bacteria make them a potential drug target.

An overwhelming majority of the TA systems encoded in the Mtb H37Rv genome are of Type II, the prominent members of which are the VapBC and MazEF families that number 50 and 10 each²⁰. Generally both the VapBC and MazEF systems are known to inhibit translation by cleaving RNAs. The toxin and antitoxin of the Type II TA systems are polypeptides. Both genes are transcribed under the same promoter. The antitoxin protein forms a complex with toxin, thereby preventing the toxic effects. Under conditions of stress, antitoxins are degraded and toxins are free to attack their cellular targets and help the cell enter a dormant state¹³. Antitoxin also represses the transcription of the operon by binding to the operator region. The toxin acts as a co-repressor at a toxin:antitoxin ratio of 1:1 but at a higher ratio, it acts as a de-repressor (Fig. 1a)²¹.

Fig. 1. NMR characterization of MazE6.

a Schematic representation of working mechanism of Type II TA system. b Sequence specific assignments are indicated on the 2D ¹H-¹⁵N HSQC spectrum of MazE6. Inset shows the assignment for the crowded region of the spectrum within the box. Assignments with a prime superscript (′) represents doubling of peaks due to conformational heterogenity. Horizontal dotted lines connect resonances of side chain amide protons. See text for details. c R₁, R_1ρ relaxation rates and steady-state heteronuclear {¹H} - ¹⁵N NOEs for ¹⁵N backbone nuclei of MazE6, plotted against residue number. Low R_1ρ relaxation rates and steady-state {¹H} - ¹⁵N heteronuclear NOE values for the C-terminal residues further confirm its disordered (dynamic) nature in solution. Blank spaces correspond to residues for which data is missing. Source data are provided in the Supplementary Data 1.

The toxins of the MazEF family in Mtb, viz., MazEF1-MazEF10 are ribonucleases and cleave specific mRNAs and rRNAs. Overexpression of MazF3, MazF6 and MazF9 has been shown to retard cell growth in M. bovis BCG and Mtb H37Rv and moreover, deletion of these genes make the bacteria more sensitive to antibiotics^22–26.

MazE antitoxins serve to neutralize MazFs and also to repress their operon as binary complexes or ternary TA-operon complexes. Analysis of sequence and available structures of MazE antitoxins have shown them to be composed of two domains: an amino-terminal DNA-binding domain (DBD) and an intrisically disordered carboxy-terminal region involved in toxin binding^27–29.

Structural data are available for MazE4, MazE7 and MazE9 from Mtb^30–32, and MazE from E. coli^33,34, B. subtilis³⁵, and S. aureus³⁶.

MazEF6 TA system plays an important role in Mtb Non Replicative Persistant (NRP) state establishment. Mtb must adapt to nutrient-limiting, hypoxic and other adverse conditions to remain dormant and persist in the host, for which the TA systems are critical. Tiwari et al. have shown that overexpression of MazF3, MazF6 and MazF9 reduced bacterial loads by 8.0-, 22.0- and 18.0-fold, respectively. The overexpression of the remaining MazF homologues, MazF1, MazF2, MazF4, MazF5, MazF7 and MazF8 did not inhibit M. bovis BCG growth. Further, the expression level of mazF6 transcripts have been shown to be even higher than those of mazF3 and mazF9 under nitrosative, hypoxic and nutrient stress conditions and upon drug treatment²⁶. Thus, the MazEF6 TA system plays a critical role in the establishment of the NRP state of Mtb.

Autoregulation of MazEF6 expression occurs by binding of the MazE6 or MazEF6 complex to its operator DNA 99 bp upstream of its gene³⁷. However, the structural, kinetic and thermodynamic factors that govern this regulation are not well characterized for MazEF6 system, or for that matter any of the MazEF systems from Mtb. The information on autoregulation of MazEF system comes from homologs of other prokaryotes like E.coli³³.

Any meaningful understanding of the TA systems requires extensive knowledge of the relationship between structure and function of this class of proteins. Therefore, it is crucial to probe the interaction of antitoxin with the operator DNA to understand the regulation of the TA system. Furthermore, the possibility of other members of Mtb MazEF family to regulate each others function is not well understood.

To this end we have investigated the structure, thermodynamic and kinetic properties of the MazE6 (residue 1–82) antitoxin protein and its interaction with operator DNA. From the solution NMR structural studies of the full length and a C-terminal deletion mutant of MazE6 (nMazE6, residue 1–49), we demonstrate that nMazE6 harbours the Ribbon-Helix-Helix (RHH) DNA binding motif. nMazE6 forms a high affinity complex with an 18 bp sequence bearing the -10 promoter region upstream of the mazE6 gene. This study provides structural and thermodynamic insights related to the operator binding mode of the protein. An Antitoxin-DNA structure has been modelled using data-driven docking protocols in HADDOCK³⁸. In addition, the potential for cross regulation of mazEF9 operon by MazE6 antitoxin has been explored. The results of these studies are presented below.

Results

Solution properties of full length antitoxin MazE6

MazE6 is highly soluble in aqueous buffers and refolds easily upon chemical denaturation. CD spectrum contains signature negative bands of both alpha helix (208 and 222 nm) and beta strand (218 nm) (Supplementary Fig. S1a). Examination of analytical size exclusion chromatogram showed that the protein has an elution volume similar to that of a 27.4 kDa globular protein (Supplementary Fig. S1c). S ize E xclusion C hromatography-M ulti A ngle L ight S cattering (SEC-MALS) confirmed that the protein is a dimer (Observed mol. wt. = 17 kDa) in solution (Supplementary Fig. S2). The one-dimensional proton NMR spectrum is resolved and has upfield shifted lines at −0.15 and −0.18 ppm suggesting that the protein is folded (Supplementary Fig. S1b). The two-dimensional ¹H - ¹⁵N HSQC NMR spectrum of free MazE6 shows several well resolved resonances with narrow line widths while the center of the spectrum (7.8–8.6 ppm) shows resonance lines of high intensity that are significantly overlapped (Fig. 1b). This suggests that the protein has a well-folded region as well as an intrinsically disordered region.

Attempts to obtain sequence specific resonance assignments on uniformly ¹³C,¹⁵N labeled proteins were unsuccessful. Hence backbone assignments were obtained from a suite of triple resonance experiments on perdeuterated protein samples that were ¹³C, ¹⁵N labeled with and without ILV-methyl protonation³⁹. The sequence specific assignments for select residues obtained from an analysis of HNCACB and HN(CO)CACB data sets are shown in Supplementary Fig. S3a. Figure 1b shows the sequence-specifically assigned ¹H-¹⁵N HSQC spectrum of MazE6. Seventy-five of the eighty-one NH resonances (82-1 proline) were assigned unambiguously in the 2D ¹H-¹⁵N HSQC spectrum of MazE6. Residues for which the assignment is missing are: M1, S27, K32, Y37, E40 and N71 . Chemical shift assignments were possible for 93.75% H^N, 70.17% ¹⁵N, 100% ¹³C^α, 75.6% CO and 98.7% ¹³C^β of the backbone and side-chain nuclei respectively. The sequence information of the protein is provided in Supplementary Fig. S1d.

The sequence specific assignments obtained from triple resonance NMR experiments were corroborated by assignment of sequential, short and medium-range NOEs observed in 3D ¹⁵N-edited NOESY-HSQC spectrum. Secondary structure of MazE6 was calculated from chemical shifts (CSI) of ¹H^α, ¹³C^α, ¹³C^β and ¹³C${}^{\prime }$, sequential and short-range NOEs, and backbone ³JH_NH_α coupling constants (Supplementary Fig. S3b). The secondary structure showed that the protein is divided into two domains: a structured N-terminal domain (M1-L46; the putative DNA binding domain) and an intrinsically disordered C-terminal domain (T47-W82; putative toxin binding domain). The extended disorder in the C-terminal region lengthens the hydrodynamic radius, causing the protein to behave like a 27.4 kDa protein in solution. The N-terminal domain is composed of one beta strand (M1-S6) and two helices (D9-E21, R26-L46) (RHH motif) that are connected by loops L1 (L7-P8) and L2 (L22-S25). The ordered and disordered nature of the N- and C-terminal domains of MazE6 also manifest in the relaxation properties of backbone ¹⁵N nuclei in the two regions. The average R₁ρ for N-terminal region is high (16.41 Hz) as compared to C-terminal region (6.13 Hz). A stark difference in the steady-state heteronulcear {¹H} - ¹⁵N NOEs for the N-terminal and C-terminal residues also shows the structured and disordered nature (Fig. 1c). The HSQC spectrum of MazE6c (G48-W82) exhibits a narrow chemical shift dispersion (Supplementary Fig. S4a) and overlays with the disorderd region of MazE6 (Supplementary Fig. S4b) providing further confirmation that the C-terminal region of MazE6 is intrinsically disordered.

Since the DNA binding region of the MazE6 is a well-folded domain, the structural studies of the interaction of MazE6 with the operator DNA were carried out using a c-terminal deletion mutant of MazE6, henceforth referred to nMazE6 (Supplementary Fig. S1d).

Solution properties of nMazE6

Like MazE6, nMazE6 exists as a stable dimer (Supplementary Fig. S5a) suggesting that the interface for homodimerization of the protein is present in this domain. The upfield shifted resonances at −0.15 ppm and −0.18 ppm are also present (Supplementary Fig. S5b) indicating that the overall fold of nMazE6 is identical to that of the same domain in MazE6. Deletion of the intrinsically disordered C-terminal domain leads to a well resolved 2D correlation spectrum (Fig. 2b) when compared to the full length protein (Supplementary Fig. S6).

Fig. 2. Solution properties of nMazE6.

a Assigned 2D ¹H-¹⁵N HSQC spectrum of nMazE6. Sequence specific assignments are indicated on the spectrum. Horizontal dotted lines connect resonances of side chain amide protons. b Strip-plots from ¹⁵N-edited NOESY data showing sequential intra-subunit and cross-strand long-range NOEs observed across the dimer interface. NOEs are colored black (intra-residue), red (intra-subunit _i−1H^α- _iH^N), cyan (inter-subunit H^α-H^N), pink (inter-subunit H^N-H^N) and green (inter-subunit side chain-H^N). β-sheet topology constructed from the pattern of assigned NOEs is shown on the side. NOEs are indicated by colored broken lines. Proposed hydrogen bonding between donor-acceptor pairs in nMazE6 are indicated by black dotted lines.

Solution structure and dynamics of nMazE6

Sequence specific resonance assignments of backbone and side chain ¹H, ¹³C and ¹⁵N nuclei were accomplished from analysis of NMR data acquired on uniformly ¹³C, ¹⁵N labeled samples of nMazE6. Chemical shift assignments were possible for 96% H^N, 92.3% ¹⁵N, 96.15% ¹³C^α, 90.38% CO and 95.9% ¹³C^β of the backbone and side-chain nuclei respectively. Also assigned were the residues that were missing in the data for the full-length protein. The ¹H, ¹⁵N and ¹³C assigned resonances of MazE6 and nMazE6 have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under accession number 51226 and 51227 respectively. The secondary structure computed for nMazE6 from ¹³C CSI and from sequential-, short- and medium-range NOEs assigned in homonuclear NOESY and isotope-edited NOESY-HSQC spectra, was found to be the same as for the N-terminal region of MazE6 i.e, a RHH (Supplementary Figs. S3b and S5c).

NOE correlations⁴⁰ were assigned in a 3D-¹³C and ¹⁵N “mother” NOESY-HSQC spectrum and two-dimensional ¹H-¹H NOESY spectra acquired at 600 MHz (τ_m = 125 ms). Structures were calculated with the understanding that nMazE6 exists in solution as a dimer. The intra-subunit restraints were duplicated and renumbered to generate restraints for both subunits. A total of 1190 unambiguous NOEs were assigned for nMazE6. Of these 1056 NOEs and 134 NOEs were assigned as intra-subunit and inter-subunit respectively. Backbone dihedral angles were assigned from a combined analysis of ¹³C secondary CSI and sequential-, short- and medium-range NOEs assigned in NOESY spectra. A total of 95 ϕ and 94 ψ dihedral angles were assigned and incorporated in the structure calculation. Knowledge of secondary structure⁴⁰ was used to generate the hydrogen bond restraints. The details of number and type of restraints used for structure calculation and the structural statistics derived for the ensemble of twenty low energy structures are given in Table 1.

Table 1.

NMR restraints and structural statistics for nMazE6.

NMR distance and dihedral constraints
Distance constraints
Totala	1190
Intra - residue (∣i − j∣ = 0)	376
Inter - residue
Sequential (∣i − j∣ 1)	322
Medium-range (∣i − j∣ ≤ 4)	194
Long-range (∣i − j∣ ≥ 5)	50
Inter-subunit	134
Hydrogen bond restraints	114
Total dihedral angle restraints	189
ϕ	95
ψ	94
Structure statistics
Violations (mean and s.d)
Distance constraints > 0.1Å	0
Dihedral angles > 5°	0
Max. Dihedral angle violation (°)	0
Max. Distance constraints violation (Å)	0
van der Waals violations	5
Deviations from idealized geometry
Bond lengths (Å)	1
Bond angles (°)	37
Impropers (°)	35
Ramachandran map statistics
PROCHECK
Most favored regions (%)	95.4
Additionally allowed regions (%)	4.6
Generously allowed regions (%)	0
Total allowed regions (%)	100
Richardson MolProbity
Favored regions (%)	98.2
Allowed regions (%)	1.1
Total allowed regions (%)	100
Disallowed regions (%)	0.7
Average pairwise r.m.s deviation Å)b
Heavy	1.0 ± 0.2
Backbone	0.44 ± 0.09

^aTotal number of restraints used for the structure calculation of the dimer.

^bPairwise r.m.s. deviation was calculated among 20 refined structures.

Each subunit of the dimeric molecule in nMazE6 structure possesses the RHH motif (β1, residues M4-L10; α1, residues D12-E24; and α2, residues R29-L49). A superposition of 20 lowest-energy conformers is shown in Fig. 3a. For the residues 5–50 and 5′–50′, average backbone and average heavy atom RMSD to mean is 0.44 and 1.0 respectively. Good stereochemical quality of the structures is reflected from the high percentage of backbone dihedral angles lying in the most favored (95.4%) and additionally allowed regions (4.6%) of the Ramachandran map. Structural coordinates for nMazE6 have been submitted in the PDB with the accession number 7WJ0.

Fig. 3. Quaternary structure and dynamics of nMazE6.

a Ensemble of twenty lowest energy structures calculated for nMazE6 emphasizing the structural symmetry of the dimer. The structures have been superposed on backbone N, C^α and C′ atoms for residues 4–52 and 4′–52′. b Cartoon representation of the lowest energy structure. The secondary structural elements are labeled in order of sequence in each subunit. The figure was generated in PyMol using the structural coordinates of nMazE6. c Alignment of MazE homologs: nMazE6 (cyan), MazE7 (yellow), MazE4 (magenta), MazE9 (navy) and BsMazE (mauve), showing the difference between them. d CH ••• π interactions between 18Vγ${}_2^{C{H}_3}$ and 32F and 8Iδ${}_1^{C{H}_3}$ and 33′ F resulting in upfield shifted lines at −0.15 and −0.18 ppm (Supplementary Fig. S5b) respectively. The distance between the methyl groups and the aromatic ring centroid (pink) is indicated by dashed line. e Aromatic cluster formed by 40Y-32′ F-33F-33′ F-32F-40′ Y. f Structure based sequence alignment of nMazE6 with other MazE from Mtb and Bacillus subtilis MazE. PDB ID: nMazE6 (7WJ0), MazE7(6A6X), MazE4 (5XE3), MazE9 (6KYT) and BsMazE (4ME7) respectively. Structural alignment was done using the DALI server⁶⁸(g) R₁, R_1ρ relaxation rates and steady-state heteronuclear {¹H} - ¹⁵N NOEs for ¹⁵N backbone nuclei of nMazE6, plotted against residue number. Source data are provided in the Supplementary Data 1.

The quaternary structure of nMazE6 (Fig. 3b) shows that dimerization occurs through the N-terminal residues of each subunit via formation of a two-stranded anti-parallel β-sheet. Inter-strand NOEs were crucial restraints for establishing the strand registry (Fig. 2b). Two RHH motifs, one from each monomer intertwine together to form a stable domain having a two-fold symmetry. The β-sheet contains alternating hydrophilic and hydrophobic residues. Hydrophobic residues point inwards and interact with aromatic residues to form the hydrophobic core. 8I and 10L from β1, 15F, 18V and 22A from α1 and 32F, 33F, 36A and 40Y from α2 form the hydrophobic core. CH ••• π and π ••• π interactions are critical to establish this hydrophobic core. CH ••• π interactions between 18Vγ${}_2^{C{H}_3}$ and 32F and 8Iδ${}_1^{C{H}_3}$ and 33′ F results in upfield shifted lines at −0.15 and −0.18 ppm respectively (Fig. 3d). An aromatic cluster formed by residues across the interface contribute significantly to dimer formation. It has a series of edge-to-face π ••• π stacking involving residues alternating from each subunit forming a donut like aromatic cluster (Fig. 3e). The residues involved are 40Y-32′ F-33F-33′ F-32F-40′ Y, with 33F-33′ F having a face to face stacking interaction. The buried surface area and the interface area formed upon dimerization are 3431.7 and 1715.85 Ų respectively.

Figure 3g shows the measured ¹⁵N R₁ and R_1ρ relaxation rates and Steady-state Heteronuclear NOE values for nMazE6 as a function of residue number. The positive Steady-state Heteronuclear NOE values suggest that nMazE6 is well ordered in solution. The relaxation rates of residues in nMazE6 are similar to those found for residues in N-terminal region of MazE6. The similarity in the relaxation parameters indicate that the nMazE6 have the same structural and dynamic properties as that of N-terminal region of MazE6 and that nMazE6 is an independently folded “domain”. Analysis of the electrostatic surface potential (Fig. 4a) of the nMazE6 structure reveals that the solvent exposed face of the β-sheet is dominated by positive potential while the distal C-terminal tail of nMazE6 has predominantly negative potential.

Fig. 4. Interaction of nMazE6 with operator DNA.

a Side view and top view of surface charge potential of nMazE6 . The molecular surface having positive potential is shown in blue and negative potential is shown in red. Electrostatic surface was calculated and rendered using the Adaptive Poisson-Boltzmann Solver package within PyMOL. b Stacked plot of gel-filtration chromatograms of operator DNA (red), MazE6 (blue) and MazE6+DNA (1:2) (black) on Superdex-S75 analytical column (24 mL). Comparison of the chromatograms shows that Peak1 and Peak2 in chromatogram (c) corresponds to DNA and MazE6-DNA complex respectively. Absorbance axis has been offset for clarity. c 1D Proton NMR spectra of analytes in Peak 1 and Peak 2. Molecular species present in Peak 1 corresponds to DNA (presence of characteristic imino protons between 12 and 14 ppm) and in Peak 2 corresponds to DNA + protein (presence of imino between 12 and 14 ppm and indole protons at 10 ppm, respectively). Line broadening of resonance line of imino protons in Peak 2 are indicative of complex formation. Similar profiles are observed for interaction between nMazE6 and DNA (Supplementary Fig. S7a). Source data are provided in the Supplementary Data 1.

MazE6-operator binding study

The operator sites of TA systems in mycobacteria are usually centered at −10 promoter region and often overlap with the regulatory elements recognized by RNA polymerase^41,42. Therefore, a stretch of 18 bp upstream of mazEF6 Transcription Start Site (TSS)⁴³ which includes the −10 promoter region was selected to probe the protein-DNA interaction using biophysical methods (Gel-filtration, Isothermal calorimetry and NMR titration). The sequence of the sense and antisense strands are 5′ -CCG GTT ATA CTA TCT GTA-3′ and 5′ -TAC AGA TAG TAT AAC CGG-3′ respectively.

An overlay of gel filtration chromatograms of MazE6, operator DNA, and MazE6+DNA (in the ratio 1:2) are shown in Fig. 4b. Comparision of the chromatograms indicate that a complex (Peak 2) is formed which elutes at a retention volume lower than that of free DNA and free protein. NMR spectroscopy of Peak 2 demonstrates the presence of both MazE6 (presence of indole protons at 10 ppm) and DNA (presence of characteristic imino protons between 12 and 14 ppm) proving the formation of complex (Fig. 4c). The experiment was repeated with nMazE6 and a clear formation of complex is seen (Supplementary Fig. S7a) in this case too. This proves that the interaction is mediated through the N-terminal domain. Also, the electrostatic potential of the N-terminal region is positive (Fig. 4a) suggesting that the DNA binding motif lies in the N-terminal region. To show that the interaction is sequence dependent and specific to its operator sequence, the operator DNA sequence was scrambled and then used to study complex formation. The elution volumes of individual components did not change showing that complex had not formed (Supplementary Fig. S7b).

Details of antitoxin and operator DNA interaction at atomic resolution were ascertained from NMR chemical shift perturbation studies. Chemical shift mapping studies were carried out for both MazE6 (Supplementary Fig. S8a) and nMazE6 (Fig. 5a and Supplementary Fig. S8b). Changes in chemical shift were observed for several residues located in the secondary structural elements in each subunit over the range of molar ratios used for the study. With increasing concentration of DNA, residues in β1, β1′, α2 and α2′ exhibited severe line broadening, to the point of disappearance at a molar ratio of 0.2. The resonance lines of these residues reappear at a higher molar ratio. This is indicative of an intermediate to a slow intermediate exchange regime. Figure 5c and d show a plot of the magnitude change in chemical shift (Δδ ppm) as a function of primary structure for nMazE6 and MazE6, respectively. Residues in the C-terminal region of MazE6 did not show any significant chemical shift perturbation indicating that they are remote from the binding site. The specific residues involved in the interaction are mapped onto the structure (Fig. 5e).

Fig. 5. Chemical Shift Perturbation (CSP) upon operator DNA binding.

a Chemical shift perturbations observed for few residues in the ¹H- ¹⁵N HSQC (Supplementary Fig. S8b) of nMazE6 upon titration with cognate operator DNA at a 1:2 (nMazE6:DNA) molar ratio. Spectra of free nMazE6 (Black) and nMazE6 in complex with operator DNA at a protein:DNA ratio of 1:2 (Red) are shown. Changes in chemical shifts of backbone amide resonance position between the first and last titration points are indicated by black arrows. For CSP of MazE6, refer Fig. S8A. b Chemical shift perturbations of key residues observed in the ¹H- ¹⁵N HSQC of nMazE6 upon titration with mazEF6 mutated operator DNA. For full spectrum, refer Supplementary Fig. S9b. c Plot of cumulative CSP as a function of the primary structure of nMazE6. d Plot of cumulative CSP as a function of the primary structure of MazE6. Average CSP and average CSP plus one standard deviation are indicated by dotted lines (Black) and (Red), respectively. e Residues that showed above-average CSP (yellow), above-average CSP + 1SD (orange), and residues that completely broadened and hence disappeared upon DNA binding (green) are mapped on the structure of nMazE6. Source data are provided in the Supplementary Data 1.

The G-X-S/T/N motif present in the loop L2 is conserved in MazE6 as well (Fig. 3f) in the form of G-M-S. It allows the correct positioning of the N-terminus of the α2 helix⁴⁴ and helps in interaction with the DNA. To test for specificity of interaction, CSP studies were carried out using operator DNA of the mazEF9 operon. In this case, a continuous shift of resonance lines without any significant line broadening was observed, indicative of fast exhange and weak binding (Supplementary Fig. S9a). The dissociation constants were calculated from the titration using TITAN software⁴⁵. TITAN quantitatively models the titration data by numerically integrating the Bloch-McConnell equations at each titration points and directly fits the peak lineshape observed in the corresponding HSQC spectrum. Of the residues which showed chemical shift perturbation >0.05 ppm (Δδ), the shifts of 24 residues were unambiguously tracked during the course of titration and were used to calculate the kinetic and thermodynamic properties of the intermolecular interaction. K_d values thus obtained were 0.09 μM for cognate mazEF6 operator DNA and 20.6 μM for non-cognate mazEF9 operator DNA and the corresponding k_off rates being 125 and 1696 per second respectively. The calculated k_on rates are 147 × 10⁷ and 8.2 × 10⁷ M⁻¹ s⁻¹ respectively. The values of these rate constants for cognate and non-cognate interactions are tabulated in Table 2. There is excellent agreement between experimentally observed lineshapes and simulated lineshapes, confirming the reliability of the fit and the parameters derived from it (Supplementary Fig. S10).

Table 2.

Thermodynamic and kinetic parameters derived for interaction of nMazE6 with operator DNA constructs.

Thermodynamic parameters derived from ITC experiments for interaction of nMazE6 with different DNA
S. No.	Experiment	Kd (μM)	ΔG (kcal/mol)	ΔH (kcal/mol)	TΔS (kcal/mol)	n
1.	nMazE6-mazEF6 operator DNA	0.29 ± 0.04	−9.08 ± 0.22	−6.13 ± 0.15	2.95	0.93 ± 0.01
2.	nMazE6-mazEF6 operator DNA	0.22 ± 0.02	−9.24 ± 0.11	−6.49 ± 0.08	2.76	0.94 ± 0.01
3.	nMazE6- mazEF6 mutated DNA	–	–	–	–	–
4.	nMazE6-scramble DNA	–	–	–	–	–
5.	nMazE6-mazEF9 operator DNA	–	–	–	–	–

Thermodynamic and kinetic parameters derived from NMR titration experiments
	Experiment		Kd (μM)	kon (M−1 s−1)	koff (sec−1)
1.	nMazE6-mazEF6 operator DNA		0.09 ± 0.01	(147 ± 22) × 107	125 ± 4.04
2.	nMazE6-mazEF6 mutated DNA		8.59 ± 0.31	(17.30 ± 0.93) × 107	1486.44 ± 27.70
3.	nMazE6-mazEF9 operator DNA		20.60 ± 1.81	(8.2 ± 0.94) × 107	1696 ± 47.14

Thermodynamic parameters of the antitoxin and operator DNA interaction which includes equilibrium dissociation constant (K_d), enthalpy change (ΔH), entropy change (ΔS), and stoichiometry (n) were extracted from Isothermal Titration Calorimetry (ITC) data. The interaction of nMazE6 with its cognate DNA is exothermic in nature with a dissociation constant of 0.22 μM and stoichiometric value(n) of 0.93 indicating an 1:1 interaction (Fig. 6a). Table 2 shows the results obtained from ITC experiments. No heat change was observed for the interaction of nMazE6 with scrambled DNA and a very small heat change accompanied titration with non-cognate mazEF9 operator DNA for which K_d could not be determined (Fig. 6c and d), indicating that the antitoxin binding is specific to its cognate operator DNA. The results of the NMR and ITC titration studies unequivocally establish that MazE6 regulate its cognate operon via a highly specific and strong interaction.

Fig. 6. Isothermal titration calorimetry of nMazE6-operator DNA binding.

a Isothermogram of the titration of operator DNA with nMazE6 at 30 °C. An exothermic interaction was observed with a K_d of 0.22 μM. b Isothermogram of mutated operator DNA at 30 °C. c, d Isothermograms of the titration of scrambled DNA and non-cognate (mazEF9) operator DNA with nMazE6 at 30 °C respectively. The top portion of each panel shows the raw titration data, and the bottom portion of each panel shows the integrated heats fit to single-binding site isotherms after subtracting the heats of dilution (endothermic in this case) (Refer Materials and method for details). The calculated (K_d) values are shown in each panel. The concentration of the titrate (DNA) was 10 μM, while that of the titrant (nMazE6) was 100 μM. (ND- Not determined). Source data are provided in the Supplementary Data 1.

HADDOCK model of MazE6-operator complex

The quaternery structure of nMazE6 shows clear separation of electrostatic potential where the positive potential is concentrated at the N-terminus and a negative potential at the C-terminus of the protein (Fig. 4a). NMR chemical shift perturbation data clearly shows that the residues at the N-terminus are responsible for DNA binding. To gain insight into the structural mechanism by which nMazE6 binds to the operator DNA, data driven docking protocols in HADDOCK were applied³⁸. Structural restraints obtained from CSP data were input as Ambiguous Interaction Restraints. Residues of nMazE6 that showed chemical shift perturbation more than average plus one standard deviation were found to be present in the positively charged surface and were considered as active residues (Refer Materials and methods).

HADDOCK clustered 181 structures (total of 200) into 7 clusters which represents 90.0% of the water-refined models HADDOCK generated. Of the 7 clusters, Cluster 1 represented the best structures with lowest Z-score and HADDOCK score of −1.2 and −106.9 ± 1.6 a.u. respectively and a cluster size of 68. The high quality of the docked structures of the complex are reflected in the low RMSD value, large buried surface area and favorable energetics of interaction. Structural statistics for the models in Cluster 1 are given in Table 3. Structural coordinates for nMazE6-operator DNA complex have been submitted in the PDB with the accession number 7WNR.

Table 3.

Structural statistics for data-driven docking of nMazE6 and operator DNA.

Ambiguous interaction restraints
nMazE6	5,7,9,15,29,30, 5′,7′,9′,15′,29′,30′
operator DNA	5,6,7,8,9,10,11,12,25,26,27,28,29,30,31,32
Restraints for nMazE6
NOE	1190
H-bond	114
Dihedral angle	189
Structural statistics for haddock-derived structures of nMazE6-DNA complex
HADDOCK score	−106.9 ± 1.6 a.u.
Cluster Size	68
RMSD	1.8 ± 1.8 Å
Evdw	−61.4 ± 1.4 kcal/mol
EEE	−240.8 ± 11.0 kcal/mol
EDE	20.7 ± 1.1 kcal/mol
ERVE	26.6 ± 14.1 kcal/mol
BSA	1383.6 ± 103.1 Å2
Z-score	−1.2

From the structures of the complex (Fig. 7a), it is clear that the β-sheet of nMazE6 is anchored in the major groove of the DNA. Residues 5K, 5′ K, 7A and 7′ A make specific base contacts in the major groove. In addition, important electrostatic interactions (E_Elec = −240.8 kcal/mol) between the sugar-phsphate backbone and the side chains of 4M, 5K, 15F, 28S, 29R and 30S from each subunit contribute to stabilization of the complex (Fig. 7b and c). It is evident from the complex structure that NNN position in the TANNNT (TATACT) nucleotide sequence is responsible for specific base contacts with the protein. To confirm the role of NNN nucleotides, the operator DNA was mutated at these positions from 5′ -CCGGTTATACTATCTGTA-3′ to 5′ -CCGGTTAGCATATCTGTA-3′. Chemical shift perturbation study showed a shift of interaction regime from slow exchange to fast exchange (Fig. 5a, b and Supplementary Fig. S9b) and that the interaction had weakened 100 fold for the mutated operator DNA (K_d = 8.5 μM) as compared to the wild type (K_d = 0.09 μM). ITC titration study indicated a very small heat change and a K_d could not be determined (Fig. 6b) for this interaction, thereby corroborating the NMR observations.

Fig. 7. HADDOCK model of nMazE6-cognate operator DNA complex.

a Cartoon representation of the ensemble of the top three HADDOCK structures with the lowest interaction energies and lowest restraints violations. b Diagrammatic illustration of protein-DNA interactions. The sequence of the operator DNA is labeled in 5′–3′ directionality. Plausible interactions of protein residues with sugar, phosphate and major groove are represented by yellow, orange and blue colored solid lines. c Specific recognition of bases by residues in β-sheet and electrostatic interaction of residues from α2 helix with sugar-phosphate backbone of TATACT -10 motif in operator region. See text for details.

Discussion

The functional studies of mycobacterial MazEF systems suggest that MazEF6 along with other TA systems play an important role in Mtb persister cell formation²⁶. The structures of the MazEF4, MazEF7 and MazEF9^30–32 provide detailed information on the structural mechanism by which the antitoxins neutralize their cognate/non-cognate toxins. Structural data for the Toxin-substrate complex is scarce. To date, data are available only for the Toxin-substrate RNA complexes of MazF6 (PDB ID 5HK0, 5HKC, 5HK3) and MazF9 (5HJZ). Importantly, the details of the structural basis of autoregulation of Mtb MazEF has not been reported.

The present study was carried out to structurally characterize the MazE6 antitoxin polypeptide and its function as a transcriptional regulator. MazE6 is a dimer in solution and harbors an RHH domain at the N-terminus similar to those observed for the homologous antitoxins, viz., MazE4, MazE7 and MazE9 despite a low sequence identity (19%, 17% and 22% respectively) with the latter. The RHH motif was first observed in the Arc-repressor protein⁴⁶ thus establishing a unique transcription factor superfamily. Other Type II systems having the same domain are Bacillus subtilis MazE, and E. coli Relb, ParD and CcdA^47–49. Structural alignment of nMazE6 with the above mentioned antitoxins shows that L10 in the loop L1, G26-M27-S28 in the loop L2 and R29 in the helix α2 are conserved. These residues help in the optimal positioning of the helices for interaction with the DNA. The extent of stabilization that is provided by the cluster of CH ••• π and π ••• π interactions to the dimerization interface of MazE6 is not seen in any of the structures of the homologs (MazE4, MazE7 and MazE9). While, MazE4, MazE7 and MazE9 have two, one and two aromatic residues in their N-terminal domain respectively, MazE6 has four of them (Fig. 3c and f). Four aromatic residues from each monomer make a donut like aromatic cluster which stabilizes the protein and makes a sort of hydrophobic core of the protein. (Fig. 3e). Thus, the N-terminal region is structurally rigid.

On the other hand, the C-terminal region of MazE6 is intrinsically disordered in the free form, except for a small helix formed by residues E62-A65. The C-terminal region is significantly populated with disorder promoting residues (76%)⁵⁰. Apart from the limited chemical shift dispersion (Fig. 1b and Supplementary Fig. S4a), low relaxation rates and negative steady-state heteronulcear {¹H} - ¹⁵N NOEs of the residues in the C-terminal region (Fig. 1c), the absence of long-range NOEs are also indicative of the flexible nature of this region. During analysis, it was found that several residues (A65, L66, A67, L75) show conformational heterogenity which could play an importatnt role in neutralizing the toxin (Fig. 1b). Multiple conformations of the protein and high mobility of the disordered region are also seen in CcdA⁴⁹. Structures of the MazEF4, EF7 and EF9 show that the C-terminal domains of the respective antitoxins are highly ordered in the TA complexes (Fig. 3c). It is expected that the C-terminal domain of MazE6 would become ordered when it neutralizes MazF6.

Autoregulation of mazEF6 operon via feedback inhibition can occur by binding of either MazE6 or by MazEF6 TA complex, wherein the latter exerts a more stringent control^37,51. Given the pathological significance of Mtb and the role of TA systems in the pathology and virulence of the organism, the sequences upstream of TSS of 2388 annotated coding genes of Mtb were further examined by Cortes et al., to determine consensus operator sequences, if any⁴³. They have shown that 73% of the Mtb coding genes possess a conserved TANNNT -10 motif centered 7–12 bp upstream of the TSS. In this study, we have unequivocally established that the −10 upstream consensus sequence is TATACT and constitutes the operator site for Mtb MazEF6 expression and regulation. Our experimental data points to this sequence being the primary operator site, given the observation that the nMazE6 dimer binds to this operator DNA in 1:1 stoichiometry with nanomolar affinity (≈217 nM). Similarly, in the E.coli MazEF system, autoregulation occurs via binding of MazE or MazEF complex to the operator site at −10 motif except that the operator region in E. coli is palindromic in sequence^33,52.

The specificity of the interaction lies in the sequence of the protein as well as the operator DNA. The MazE proteins in Mtb are structurally conserved. Despite this structural conservation, the residues involved in DNA binding and making specific base contacts (residues in β-strand) are not conserved (Fig. 3f). Binding of MazE6 and nMazE6 to cognate operator DNA involves the same residues in the RHH motif (Fig. 5 and Supplementary Fig. S8). In the case of MazE6, our data has shown that C-terminal domain does not interact with the DNA. The residues important for protein-DNA interaction are those present in the β-sheet and S28-S30 in helix α2. Similar results were observed by Zhao et al., where they found that S25A and R26A mutations abolish DNA binding³⁷. Structure of AtaR-AtaT-DNA complex support a similar method of interaction where residues in β-strand provide specificty and residues at the start of α2-helix anchor the protein on the DNA⁵³. Specificity of interaction lies in the operator DNA sequence as well. For instance, the operator DNA for mazEF6 (TATACT) and for mazEF9 (TAGCAT) differ significantly in the type of nucleotide (purine or pyrimidine) present at the positions NNN in the consensus TANNNT sequence. Apart from the differences in the TANNNT sequence, mazEF6 operator DNA has an additional GGT sequence upstream of TANNNT, while mazEF9 operator DNA has TGG. These additional sequences are known to influence the promoter activity to different strength⁴³. Mutation of the NNN nucleotide in the cognate DNA results in loss of interaction. This strongly suggests that the -10 promoter region is the high affinity and specific site and might be the nucleation site for mazEF6 transcription autoregulation. These results are similar to those found for ccdAB⁴⁹ and mazEF system³³ from E.coli where the RHH domain of antitoxin CcdA and AbrB domain of MazE interacts with a high affinity operator site bearing the -10 promoter region.

A good agreement between the interaction studies carried out by ITC and NMR titration is observed (Table 2). A very small heat change was observed in ITC for interaction with non-cognate mazEF9 operator DNA, while NMR titration studies showed very fast exchange without any line broadening. The observations from the ITC titrations are strongly supported by detailed lineshape analysis carried out using the modified Bloch-McConnell equations in TITAN. In the case of the interaction of nMazE6 with mazEF9 operator the k_on is almost 18 times slower while k_off is ~14 times faster resulting in a significantly shorter life time for the complex as reflected in the much higher K_d. These studies have shown that MazE6 interacts with its operator DNA to regulate its transcription and is unlikely to interact with non-cognate mazEF9 operator in-vivo.

Our studies have shown that in MazE6, the N-terminal DNA binding domain is highly structured, whereas the C-terminal Toxin binding domain is intrinsically disordered. The studies have shown that MazE6 and nMazE6 bind to the cognate operon with high affinity. The high resolution NMR studies have identified the key residues neccessary for this binding. Furthermore, we have shown that the MazE6 does not bind to the mazEF9 operator DNA. Hence, it is safe to say that MazE6 is unlikely to regulate the transcription of mazEF9 operon. The weak interaction between nMazE6 and mazEF9 operator DNA in in vitro condition might be a reflection of the non-specific electrostatic interactions between the backbone of the non-cognate DNA and the RHH domain of nMazE6⁵⁴. Mtb is one of the most lethal pathogenic bacteria which claims nearly 2 million lives per year worldwide⁵⁵. The MazEF TA systems in Mtb are important for establishing the "Non Replicative Peristent” state. It has been observed that 70% of Mtb’s total RNA is susceptible to MazF6 RNase activity⁵⁶. Mechanisms that hinder the MazE6-operator interaction will potentially disrupt the repression of transcription leading to increased levels of toxin activity as an ‘intracellular death factor’. Synthetic oligonucleotides or other ligands that will compete for the DNA recognition motif or ligands that bind to other regions of MazE6 which will change its ability to interact with operator DNA can be designed based on the above study. The structure and dynamics of MazE6 and its’ interaction with the operator DNA described above improves our understanding of the structural basis for this regulation and will aid in the rational design of inhibitors against Mtb.

Materials and methods

Protein expression and purification

MazE6 protein was co-expressed with hexa-histidine tagged MazF6 (Toxin) from pETDuet-1 vector from MCS2 and MCS1 respectively using E. coli BL21(DE3) overexpression strains. Cells were grown at 37 °C with 1% primary inoculum. Induction of protein expression was carried out by addition of IPTG to a final concentration of 0.5 mM to the culture broth when the culture reached an O.D₆₀₀ 0.8. Cells were harvested after 16 h of induction at 20 °C by centrifugation (30 min/6000 rpm/4 °C) and resuspended in 50 mM Tris, 500 mM NaCl, 20 mM Imidazole, pH 8.0. The suspension was again centrifuged (30 min/13,000 rpm/4 °C) after lysing the cell by French Press at 1200 psi. Cell lysate containing MazEF6 complex was loaded on Ni-NTA affinity column. Denaturation buffer (50 mM Tris, 500 mM NaCl, 4M Guanidine Hydrochloride, pH 8.0) was then passed to separate MazF6 from MazE6. MazE6 was collected in flowthrough during denaturaion step. The bound MazF6 protein was eluted with a gradient of 0–1 M Imidazole. MazE6 was refolded by dialysing the protein in buffer comprising of 50 mM Tris, 300 mM NaCl, 50 mM Arginine Hydrochloride and 10% Glycerol at pH 8.0. Final gel filtration step was performed against 20 mM phosphate buffer containing 20 mM NaCl, 1 mM EDTA, pH 7.0.

For nMazE6, the pET-21a(+)-nmazE6 vector with a hexa-histidine tag and TEV cleavage site at the N-terminus was transformed into E. coli BL21(DE3) overexpression strains. Cells were grown at 37 °C with 1% primary inoculum. Induction of protein expression was carried out by addition of IPTG to a final concentration of 0.5 mM to the culture broth when the culture reached an O.D₆₀₀ 0.8. Cells were harvested after 16 h of induction at 20 °C by centrifugation (30 min/6000 rpm/4 °C) and resuspended in 50 mM Tris, 500 mM NaCl, 20 mM Imidazole, pH 8.0. The suspension was again centrifuged (30 min/13,000 rpm/4 °C) after lysing the cell by French Press at 1200 psi. Purification was carried out using Ni-NTA affinity chromatography. Purified fusion protein was then dialyzed into TEV cleavage buffer (50 mM Tris, 250 mM NaCl, pH 8.0) and digested with TEV protease. The reaction products were separated by Ni-NTA affinity chromatography. Final step of gel filtration was performed against 20 mM Potassium Phosphate, 20 mM NaCl, 1 mM EDTA, pH 7.0.

For MazE6c, the pET-21a(+)-mazE6c vector with a hexa-histidine tag followed by Cytochrome b₅ tag at the N-terminus was transformed into E. coli BL21(DE3) overexpression strains. Cells were grown at 37 °C with 1% primary inoculum. Induction of protein expression was carried out by addition of IPTG to a final concentration of 0.5 mM to the culture broth when the culture reached an O.D₆₀₀ 0.8. Cells were harvested after 5 h of induction at 37 °C by centrifugation (30 min/6000 rpm/4 °C) and resuspended in 50 mM Tris, 500 mM NaCl, 20 mM Imidazole, pH 8.0. Purification was carried out using Ni-NTA affinity chromatography. Purified fusion protein was then dialyzed into thrombin cleavage buffer (50 mM Tris, 100 mM NaCl, 200 mM CaCl₂, pH 8.0) and digested with thrombin (Sigma Chemicals Ltd.). The reaction products were separated by gel filtration against appropriate NMR sample buffer.

Stable isotope precursors for protein production were purchased from Sigma Chemicals or from Cambridge Isotope Laboratories. Protein samples that were isotopically enriched in ¹⁵N or ¹³C,¹⁵N were prepared by culturing cells bearing the appropriate plasmids in minimal media containing ¹⁵NH₄Cl or ¹⁵NH₄Cl and ¹³C₆ - glucose as the sole sources of nitrogen and carbon dissolved in H₂O⁵⁷. Protein samples that were enriched in ²H, ¹³C and ¹⁵N were prepared by culturing cells in minimal media containing ²H₇,¹³C₆ - glucose, and ¹⁵NH₄Cl as carbon and nitrogen sources respectively, dissolved in 99% enriched D₂O. ILV-methyl protonated samples were prepared using ²H₇,¹³C₆ - glucose and ¹⁵NH₄Cl as carbon and nitrogen sources respectively, supplemented with 3,3-²H₂, ¹³C₄ - α-ketobutyric acid and 3-²H,¹³C₅ - α-ketoisovaleric acid⁵⁸, in 99% D₂O as solvent.

NMR sample preparation

NMR samples of MazE6, nMazE6 and MazE6c were prepared by dissolving protein in 90% aqueous buffer (20 mM KPi pH 7.0, 50 mM NaCl, 1 mM EDTA) and 10% D₂O, except for the 3D HCCHTOCSY(100% D₂O). The concentration of proteins for all experiments ranged from 300 to 600 μM except for titraton experiments, where the initial protein concentraion was set to 100 μM. Chemical shifts were referenced to external DSS.

NMR data acquisition

All spectra for assignment and structure calculation were acquired on an Agilent (Varian) DDS2 600 MHz spectrometer equipped with a cryogenically cooled cold-probe fitted with a pulsed field gradient (z-axis only) accessory at 30 °C for MazE6 and at 25 °C for nMazE6 and MazE6c.

NMR data processing and analysis

NMR data were processed on Intel workstations running on CentOS 7 operating systems using NMRPipe/NMRDraw processing software⁵⁹. Spectra were analyzed using the CCPNMR (versions 2.1–2.4) suite of programs ^ccpn.Backbone atoms of the proteins were assigned using HNCACB, CBCA(CO)NH (HN(CO)CACB for MazE), HNCO and HN(CA)CO. Aliphatic side-chains were assigned using correlations from C(CO)NH and HC(CO)NH . For aromatic side chains, ¹H-¹H TOCSY, ¹H-¹H NOESY, ¹³C-HSQC and ¹³C-NOESY-HSQC spectra were used.

nMazE6 structure calculation

Structures of nMazE6 were calculated by CYANA version 2.1⁶⁰ using experimentally determined distance and dihedral angle restraints. Unambiguously assigned NOE correlations from ¹³C-NOESY-HSQC, ¹⁵N-NOESY-HSQC, ¹H-¹H NOESY spectra were used to derive distance restraints from their peak intensities. A lower bound of 1.8 Å was set for all distance restraints. To generate hydogen bond restraints, knowledge of secondary structure was used. Upper distance bounds of 2.2 Å for NH ⋯ O bonds and 3.2 Å for N ⋯ O bonds were set as restraints between hydrogen bonded donor and acceptor pairs. Backbone dihedral angles were computed by DANGLE, a program embedded within CCPNMR⁶¹, from ¹³C^α, ¹³C^β and ¹³C’ secondary CSI. During structure calculation, 999 random conformers were subjected to 10,000 steps of annealing to obtain an ensemble of 50 structures of acceptable stereochemical quality. Final structural refinement with water was done by XPLOR-NIH⁶² with output files from CYANA. First, CYANA structures were converted into the XPLOR format using pdbstat⁶³ with a 10% range for the upper bound (upper limit for NOEs), and the van Der Waals radius was used as the lower bound (lower limit for NOEs). The protein structure validation suite wwPDB Validation server⁶⁴ has been used to check the quality of the structure. The twenty lowest energy structures were used for further analysis.

MazE6-operator binding study by Isothermal Titration Calorimetry (ITC)

A stretch of 18 bp upstream of mazEF6 TSS which invloves −10 promoter region was taken for interaction study named as mazEF6 operator DNA. ITC experiments were carried out using MicroCal VP-ITC instrument (GE) at 303 K. Samples were degassed before the experiment. For the titrations, 10–30 μM of the DNA was used in sample cell and was titrated with 100–300 μM of nMazE6 respectively. Thirty injections of the titrant were performed at an interval of 180 s. Interaction studies of nMazE6 with mazEF6 mutated operator DNA and mazEF9 operator DNA were done at a concentration of 10 μM DNA in sample cell and 100 μM nMazE6 in syringe. A control experiment was done with scrambled DNA having same nucleotide base pairs as the cognate DNA but with scrambled sequnce. All the DNA sequences used in the study are listed in Table S1. For heats of dilution, separate protein-in-buffer experiments were performed and subtracted from the integrated heat and the data were fit for a one site-binding model. All the parameters were kept floating during data fitting. The experiments were repeated twice or thrice for consistency.

Chemical shift mapping of MazE6-operator binding

Binding of antitoxin to its cognate mazEF6 operator DNA, mazEF6 mutated operator DNA and non-cognate mazEF9 operator DNA was also probed by chemical shift mapping of MazE6 and nMazE6 upon addition of DNA. Either 50 or 100 μM ¹⁵N- labeled protein (MazE6 or nMazE6) was titrated with unlabeled DNA ranging from 0 to 100 or 200 μ M at 298 K.

To determine the combined and weighted backbone amide chemical shift perturbation (Δδ) was calculated using the standard formula:

$$\mathrm{\Delta }\delta ={\left[{\left(\mathrm{\Delta }{\delta }_H\right)}^2+{\left(\mathrm{\Delta }{\delta }_N/5\right)}^2\right]}^{1/2}$$ 1

where Δδ_H and Δδ_N are the chemical shift changes (in ppm) at the beginning and the end of the titration.

NMR titration fitting by TITAN

Two dimensional lineshape analysis of NMR titration data was done by TITAN software package. It numerically simulates the evolution of magnetization during a pulse sequence in the presence of chemical exchange, in the Liouville space. All the ¹H- ¹⁵N- HSQC spectra of a titration series were processed identically. Two-state binding model was used to fit the following twenty-three residues of nMazE6 showing perturbation in the chemical shift: 4M, 5K, 7A, 12D, 13E, 14T, 15F, 17R, 18V, 19S, 20R, 22A, 23S, 25L, 26G, 27M, 32F, 36A, 39R, 41L, 42H, 44L and 46A.

¹⁵N-backbone dynamics

A series of 2D ¹H-¹⁵N HSQC spectra with different T₁ and T_1ρ relaxation delay time intervals were acquired to measure spin-lattice relaxation rate R₁ (1/T₁), and spin-spin relaxation rate R_1ρ (1/T_1ρ) respectively. Each t₁ data time point was signal averaged over 32 transients. The pulse schemes described by Farrow et al. was used to acquire the data sets⁶⁵. For measuring T₁, relaxation delays were set to 10, 50, 100, 200, 400, 600, 800, 1000, 1100, 1300, and 1500 ms. For T₁ρ the relaxation delays were set to 2, 5, 10, 20, 30, 40, 50, 60, 70, and 80 ms. ¹H-¹⁵N Heteronuclear NOEs were measured from the ratio of peak intensities (I_on/I_off) with and without the saturation of the amide protons for 3 s.

Structure calculation of nMazE6-operator complex

Data driven structural model of the complex was generated by HADDOCK 2.4. The ensemble of 20 lowest energy structure of nMazE6 was used for docking on the energy minimized 3D model of operator DNA generated by Web 3DNA (w3DNA) 2.0 server⁶⁶. The residues showing Chemical Shift Perturbation (CSP) more than the average perturbation were investigated for their location in the structure and accessiblity. The active residues defined in protein for docking were: 5K, 7A, 9S, 15F, 29R, 30S, 5K′, 7A′, 9S′, 15F′, 29R′, 30S′ . For DNA, ‘all nucleotides’ were considered active in initial runs. The nucleotides to which the initial runs pointed to, were taken as active residues in final run : 5–12 and 25–32. The docked structures with the lowest intermolecular energies given as output from HADDOCK were analyzed using PyMOL⁶⁷.

Statistics and reproducibility

All ITC experiments described were performed in independent duplicates. NMR titration for nMazE6-cognate operator DNA was performed in independent duplicates.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Author contributions

Project was concieved by S.P.S. and K.K. NMR data acquisition and analysis was done by K.K. and S.P.S. ITC data was acquired by K.K. and analyzed by K.K. and S.P.S. HADDOCK runs were performed by K.K. and analyzed by K.K. and S.P.S.

Peer review

Peer review information

 Communications Biology thanks Cameron Mackereth, Ralph Bertram and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Joanna Timmins, Christina Karlsson Rosenthal, and Gene Chong. Peer reviewer reports are available.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. The ¹H, ¹³C and ¹⁵N assigned resonances of MazE6 and nMazE6 have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu/) under accession number 51226 and 51227 respectively. The structural coordinates and experimentally derived restraints for nMazE6 and nMazE6-operator DNA complex have been deposited in the Protein Data Bank with accession number 7WJ0 and 7WNR respectively. Source data are provided with this paper (Supplementary Data 1).

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-022-03933-5.