The helicase–primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase–primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB–DnaG complex. Also, MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae. The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.

Introduction

DNA replication is a fundamental process in all domains of life and requires a large molecular machine coordinating the action of dozens of discrete factors to ensure accurate genome inheritance [1]. The interaction between the bacterial replicative ring helicase (DnaB) and the primase (DnaG) is essential for the formation of the functional primosome at oriC to initiate the DNA replication [2,3]. DnaB helicase unwinds duplex DNA into single-stranded DNA (ssDNA), with this unwinding fueled by the hydrolysis of nucleoside triphosphate at the replication fork [46], and DnaG primase uses the newly formed ssDNA as a template for the de novo synthesis of RNA primers to initiate the DNA replication [7]. The interaction between DnaB and DnaG mutually stimulates both of their activities [8]. DnaG increases both the NTPase and helicase activities of DnaB [9], while DnaB increases and modulates the synthesis of RNA primers by DnaG [7,10,11]. The DnaG primase consists of three domains with distinct activities: an N-terminal zinc-binding domain, a central RNA polymerase domain and a C-terminal helicase-binding domain. The C-terminal domain of DnaG (DnaG-CTD) interacts with the N-terminal domain of DnaB (DnaB-NTD) and stimulates the DnaB helicase activity [12]. The stability of the interaction between DnaG primase and DnaB helicase varies among eubacteria. The helicase–primase interaction has been reported to be weak in Escherichia coli [13], moderate in Helicobacter pylori [14,15] and strong in Bacillus stearothermophilus [9]. Despite its relatively low resolution, the only available structure of the complex of DnaG-CTD with DnaB helicase in B. stearothermophilus did provide insights into the helicase–primase interaction pattern [16]. Since the helicase-binding domain of primases is poorly conserved, we expect the structures from different organisms to provide insights into the mode of interaction between DnaG and DnaB.

DNA replication in Mycobacterium tuberculosis (Mt) is not well understood. This intracellular pathogen is the main cause of human tuberculosis [17], and according to the recent World Health Organisation report (WHO report 2017), tuberculosis is the leading cause of death from a single infectious agent. This pathogen can, under favorable conditions, hide in a dormant state in the host for long periods of time without producing conspicuous disease symptoms [18]. Specifically, M. tuberculosis progresses to a state of non-replicating persistence to lie dormant in the host for such long periods of time [19]. Mutations generated by error-prone DNA replication also enhance M. tuberculosis virulence and lead to antibiotic resistance [20]. Part of our lack of understanding of DNA replication in M. tuberculosis arises from the homologs of some essential proteins for efficient DNA replication in E. coli having not been identified in mycobacteria. In addition, the regulators of DnaA activity (Hda in E. coli and YabA in B. subtilis), replicative helicase loader protein (DnaC in E. coli and DnaI in B. subtilis) and replication terminator proteins (Tus in E. coli and RTP in B. subtilis) have not been characterized in M. tuberculosis [2124]. Recently, Rv0004 was found to have a role in M. tuberculosis DNA replication; it interacts with DNA and the DnaB [25]. The mechanism, by which the replisome of M. tuberculosis is formed from the DnaG–DnaB complex and various accessory proteins, is still not clear. M. tuberculosis DnaB (MtDnaB) has recently been functionally characterized [26]. The MtdnaB gene encodes 874 amino acid residues, 416 of which (from Leu400 to Asn815) form an intein [27]. An intein is an internal segment of amino acids that are excised from a protein precursor to generate a functional protein that splits off from the main protein during post-translational maturation [27]. The lack of structural data for the DnaB–DnaG complex and the incomplete structural information for the individual proteins are obstructing our efforts to understand the molecular details that reinforce this essential interaction in M. tuberculosis. However, the crystal structure of the NTD of MtDnaB has been reported [28]. To further study the mode of interaction between MtDnaB and MtDnaG, we determined the structure of the helicase-binding domain of MtDnaG.

The MtDnaG-CTD structure was observed to differ from the other available DnaG-CTD structures. We used crystal structures of MtDnaB-NTD reported earlier [28] and the currently determined structure of MtDnaG-CTD to derive a model of helicase–primase interactions in M. tuberculosis, and provided further evidence for this model by performing in vitro experiments. We identified single amino acid residues at the helicase-binding domain of MtDnaG that, when mutated to alanine, resulted in proteins with altered interactions with DnaB helicase. Further analysis revealed the possibility of cross-species interaction of MtDnaB with other DnaG in eubacteria. Overall, these results substantially helped us to understand the mechanism of helicase–primase interactions specifically in M. tuberculosis and expand our knowledge on the diversity of bacterial DNA replication strategies, some of which may be organism specific.

Materials and methods

Cloning of expression constructs for heterologous expression

Mycobacterium tuberculosis (H37rv) genomic DNA and specific primers (Supplementary Table S1) were used for the polymerase chain reaction (PCR) to amplify the coding regions of MtdnaG-CTD (477 base pairs) and MtdnaB-NTD (591 base pairs). The amplified products of MtdnaG-CTD and MtdnaB-NTD were cloned into the Nhe1/Xho1 site of the expression vector pET21c (Novagen, Madison, WI, U.S.A.) and the Nde1/Xho1 site of the expression vector pET28b (Novagen, Madison, WI, U.S.A.), with each containing the C-terminal His6 tag. Similarly, dnaG-CTD from Vibrio cholerae (O395 strain) was cloned into the Nco1/Xho1 site of pET28b (Rehman thesis). DNA sequencing confirmed the sequences of all constructs. The cloning of HpdnaG-CTD and EcdnaG-CTD was performed previously in our laboratory [15,29].

Site-directed mutagenesis

MtDnaG-CTD mutants (MtDnaG-CTD I605A and MtDnaG-CTD F615A) were prepared by following a PCR-based method using the pET21c expression vector encoding for the wild-type (MtDnaG-CTD) protein as a template. The amplification of the expression vector was performed using the KOD hot-start DNA polymerase (EMD Millipore), following the manufacturer's guidelines, and specific pairs of primers (Supplementary Table S1) were used for the introduction of the desired mutation. PCR products were treated with DpnI enzyme (New England Biolabs) and transformed into DH5α E. coli cells. The mutations were confirmed by performing DNA sequencing.

Overexpression and purification of recombinant proteins

pET21c-MtDnaG-CTD and point mutant (MtDnaG-CTD I605A and MtDnaG-CTD F615A) constructs were transformed into E. coli BL21 (DE3). For expression, 1% of the overnight-grown culture of a single colony was added to 2 l of Luria-Bertani (LB) broth containing 100 µg/ml ampicillin, 1 mM glucose and 1 mM MgCl2. Cells were grown to an optical density of 0.6 (in 600 nm wavelength) at 37°C. Overexpression of protein was induced overnight at 16°C with 0.5 mM isopropyl-beta-d-thiogalactopyranoside (IPTG). The cells were harvested at 8000 rpm for 5 min and mixed with lysis buffer A (30 mM Tris–HCl [pH 7.5], 150 mM NaCl, 5 mM imidazole, 5 mM MgCl2, 5 mM β-mercaptoethanol [βME], 100 µM phenylmethyl sulfonyl fluoride [PMSF]). After the addition of lysozyme (0.1 mg/ml), the cell suspension was incubated at 4°C for 30 min. The cell suspension was sonicated (Branson Ultrasonic Systems) in an ice–water mixture at 25% of the amplitude with a pulse of 20 s each interspersed with an interval of 30 s, and cell lysate was treated with 0.1% Triton X-100, followed by incubation for 30 min on a rotating rocker at 4°C. The cell lysate was cleared by being subjected to centrifugation at 13 000 rpm for 45 min at 4°C and then purified using Ni-NTA Sepharose resin (GE Healthcare, Sweden) pre-equilibrated in buffer A. Proteins were eluted with buffer B (30 mM Tris–Cl [pH 7.5], 150 mM NaCl, 250 mM imidazole, 5 mM βME and 100 µM PMSF). The protein was then concentrated using an AmiconUltra-4 centrifugal filter ultracel-10K (Millipore, Billerica, MA, U.S.A.) and purified further by using a gel filtration chromatography Hi-Load Superdex-75 16/60 column (GE Healthcare) pre-equilibrated with buffer C (30 mM Tris–Cl [pH 7.5], 150 mM NaCl, 5% glycerol and 5 mM β-ME). Peak fractions were checked using SDS–12% PAGE and pooled together.

HpDnaG-CTD and EcDnaG-CTD were purified as described earlier [15,29]. MtDnaB-NTD and VcDnaG-CTD constructs were transformed into E. coli BL21 (DE3) cells. Cells containing plasmids were grown in LB media in the presence of either 50 µg/ml kanamycin (MtDnaB-NTD) or 100 µg/ml ampicillin (VcDnaG-CTD) up to an OD600 of 0.6–0.8 at 37°C. Overexpression of protein was induced overnight at 20°C with 0.2 mM IPTG. Bacterial cell pellets were resuspended in lysis buffer A and cells were disrupted by subjecting them to sonication. The lysate was cleared by subjecting them to centrifugation at 13 000g for 45 min at 4°C; and the proteins here were purified by using Ni-NTA Sepharose beads pre-equilibrated in buffer A, and eluted with buffer B. Final purification was done using gel filtration on a Hi-Load Superdex-75 16/60 column pre-equilibrated with buffer G (20 mM HEPES [pH 7.4] and 150 mM NaCl). The homogeneity of protein was checked with (12%) SDS–PAGE.

Overexpression and purification of the selenomethionine-labeled MtDnaG-CTD

Selenomethionine (Se-Met)-labeled MtDnaG-CTD was prepared to provide phases necessary for structure determination. Se-Met-labeled protein was purified under reducing conditions to prevent the formation of a mixture of reduced and oxidized Se-Met-labeled proteins. Protein labeling was carried out using media supplied by Molecular Dimensions. The concentration of selenomethionine was maintained at ∼25 mg/l. Initially, the primary culture of MtDnaG-CTD-transformed E. coli BL21 (DE3) was grown in LB medium overnight. The next morning, cells were harvested by centrifuging the overnight grown culture at 4000 rpm for 5 min. The pellet obtained was resuspended in a purely selenomethionine medium and the process was repeated once again to remove any traces of LB medium. After inoculation of the secondary culture in complete selenomethionine medium, the culture was allowed to grow for 4 h until the OD600 reached 0.5 at 37°C. After this procedure, the cells were induced with 1 mM IPTG, and then shaken for another 8 h at 30°C, harvested at 8000 rpm for 3 min and stored at −80°C for downstream processing. The remaining steps of the purification were similar to those followed for the native MtDnaG-CTD, as described above.

Crystallization of MtDnaG-CTD

Native MtDnaG-CTD (6 mg/ml) and selenomethionine-labeled MtDnaG-CTD (5 mg/ml) were subjected to extensive crystallization trials using the hanging drop method. Initially, the automated machine MOSQUITO™ (AIRF, JNU) was used to set hanging crystallization drops containing 500 nl protein solution plus 500 nl reservoir solution in 96-well Greiner-CELLSTAR plates for screening with Hampton and Molecular Dimensions crystallization screens. Small protein needles were grown using the reservoir solution of Hampton Peg Ion screen 25 (20% (w/v) PEG 3350, 0.2 M magnesium acetate tetrahydrate). The conditions were replicated and improved in 24-well Linbro plates by mixing 2 µl protein solution with 2 µl reservoir solution and equilibrating the resulting mixture against a 500 ml reservoir solution at 4°C. The best selenomethionine-labeled protein crystals were grown with a reservoir solution of 18% (w/v) PEG 3350, 0.15 M magnesium acetate and MOPS buffer pH 7.6. Crystals appeared after 1 week and grew to full size within 2 weeks.

Data collection, processing and structure determination

The crystals were flash-frozen in liquid nitrogen with a cryoprotectant solution containing 20% PEG 400 mixed with mother liquor (18% (w/v) PEG 3350, 0.15 M magnesium acetate and MOPS buffer with pH 7.6). The X-ray data for Se-Met-labeled crystals were collected at the BM14 synchrotron beamline (ESRF, Grenoble, France) at a selenium peak wavelength of 0.9786 Å. The data sets were indexed, integrated and scaled using HKL-2000 data processing software [30]. The input diffraction data were prepared using programs of the CCP4 suite [31,32]. A partial structure was solved using the single-wavelength anomalous scattering protocol of Auto-Rickshaw of the EMBL-Hamburg automated crystal structure determination platform [33]. Anomalous data were used to calculate FA values using the program SHELXC [34]. Based on an initial analysis of the data, the maximum resolution for substructure determination and initial phase calculation was set to 2.0 Å. All of the three heavy atoms expected (three selenomethionine atoms for each molecule of the asymmetric unit) were found using the program SHELXD [35]. The correct hand for the substructure was determined using the programs ABS [36] and SHELXE [37]. Initial phases were calculated after density modification using SHELXE. The initial phases were improved by carrying out density modification and phase extension to a resolution of 1.57 Å using the program RESOLVE [38]. The initial model was built using the auto-build program ARP/wARP [39]. The regions missing from this model were built manually using the COOT graphics package [40], and refinement of the resulting model was carried out with REFMAC5 [41]. The structure was further improved after the iterative model building and refinement using COOT [42] and REFMAC5 [43]. The final crystal structure was well refined with excellent electron density and validated by using PROCHECK [44] of the CCP4 suite. The figures presented here were generated using PyMOL [45]. The data collection and refinement statistics are given in Table 1. Co-ordinate and structure factor files for the crystals of MtDnaG-CTD have been deposited with the Protein Data Bank under accession code PDB ID 5Z51.

Table 1
Crystallographic data and refinement statistics
Crystallographic data 
 X-ray source BM14, ESRF, France 
 Wavelength (Å) 0.97 
 Space group P2121
Unit cell parameters 
a, b, c (Å) 48.28, 137.55, 36.51 
α, β, γ (°) 90, 90, 90 
 Resolution range (Å) 68.78–1.58 
 Completeness (%) 92.8 (99.8) 
 Total no. of observations 150 161 
 No. of unique observations 40 035 
 Redundancy 3.8 (3.9) 
 Average I/σ (I19.66 (1.94) 
CC (1/2) 0.92 (0.72) 
 Rr.i.m. 0.088 
 Crystal mosaicity (°) 0.76 
Refinement 
R-factor (%) 18.14 
 Free R-factor (%) 20.88 
 Mean B-factor (Å216.1 
 Number of atoms 
 Protein/PEG/acetate/water 1812/17/7/320 
RMS deviation 
 Bond length (Å) 0.017 
 Bond angles (°) 1.64 
 Ramachandran plot (%) 
 In preferred region 99.1 
 In allowed region 0.5 
 Outliers 0.4 
 PDB ID 5Z51 
Crystallographic data 
 X-ray source BM14, ESRF, France 
 Wavelength (Å) 0.97 
 Space group P2121
Unit cell parameters 
a, b, c (Å) 48.28, 137.55, 36.51 
α, β, γ (°) 90, 90, 90 
 Resolution range (Å) 68.78–1.58 
 Completeness (%) 92.8 (99.8) 
 Total no. of observations 150 161 
 No. of unique observations 40 035 
 Redundancy 3.8 (3.9) 
 Average I/σ (I19.66 (1.94) 
CC (1/2) 0.92 (0.72) 
 Rr.i.m. 0.088 
 Crystal mosaicity (°) 0.76 
Refinement 
R-factor (%) 18.14 
 Free R-factor (%) 20.88 
 Mean B-factor (Å216.1 
 Number of atoms 
 Protein/PEG/acetate/water 1812/17/7/320 
RMS deviation 
 Bond length (Å) 0.017 
 Bond angles (°) 1.64 
 Ramachandran plot (%) 
 In preferred region 99.1 
 In allowed region 0.5 
 Outliers 0.4 
 PDB ID 5Z51 

Construction of the helicase–primase complex model in M. tuberculosis

The structure of MtDnaB-NTD reported earlier by the Tsodikov group [28] and our current structure of MtDnaG-CTD, as well as the structure of the BstDnaB–BstDnaG-CTD (helicase–primase) complex [16] as a template, were used to model the MtDnaB-NTD/MtDnaG-CTD (helicase–primase) complex of M. tuberculosis. The structure of MtDnaB-NTD was superimposed on that of BstDnaB-NTD, and the globular region (GR) of MtDnaG-CTD was superimposed on the GR of BstDnaG-CTD of the complex. To superimpose the helical hairpin region (HHR) of MtDnaG-CTD with the HHR of the BstDnaB–BstDnaG-CTD complex, the HHR of MtDnaG-CTD was rotated by ∼32°, keeping the linker between the HHR and the GR as the center of rotation, to obtain the lowest root-mean-square deviation (RMSD) between the BstDnaG-CTD and MtDnaG-CTD structures. This modification maximized the interactions between primase and helicase. The structure of the modeled complex was relaxed, by carrying out molecular energy minimization using the GROMACS molecular dynamics package [46], in order to eliminate bad contacts. Similarly, we constructed models of three complexes of MtDnaB-NTD, one with EcDnaG-CTD, and the other two with VcDnaG-CTD and HpDnaG-CTD, respectively, to study the possible interactions and stability levels of the heterocomplex.

Molecular dynamic simulation for binding affinity estimation

Molecular dynamic simulations were performed on the models of the MtDnaB-NTD complex with DnaG-CTD, DnaG-CTD I605A and DnaG-CTD F615A, using the GROMACS 5.1.4 package, with the GROMOS 96 force field [47]. To create the in silico mutants, isoleucine 605 and phenylalanine 615 were each changed to alanine using PyMOL [45] to make the two different DnaG-CTD mutants. These proteins were each solvated in a dodecahedron solvation box by using a simple point charge [48] of water molecules. Na+ and Cl ions were included to neutralize the system. The energy was minimized using 500 steps of steepest descent, followed by 1000 steps of conjugate gradient. For maintaining the system in a stable environment (300 K, 1 bar), Berendsen temperature coupling [49] and Parrinello-Rahman pressure coupling [50] were employed and were set to 0.1 and 2.0 ps for temperature and pressure, respectively. A particle mesh Ewald algorithm [51] was run to measure the electrostatic and van der Waals interactions; here, the cutoff distance for short-range VdW (rvdw) interactions was set to 1.4 nm, and Coulomb cutoff (r coulomb) and a neighbor list (rlist) was set to 0.9 nm. The LINCS algorithm [52] was used to measure all of the constrained bond lengths and the time step was set to 0.002 ps. The complexes in a medium were equilibrated for 100 ps in NPT and NVT ensembles. Finally, a 25 ns molecular dynamic simulation was carried out for each tested structure. All trajectories were stored every 2 ps for further analysis. Similar molecular dynamic simulation steps, as described above, were used on the three models of the complexes of MtDnaB-NTD, i.e. with EcDnaG-CTD, HpDnaG-CTD and VcDnaG-CTD. The binding free energies were calculated by using the molecular mechanics Poisson–Boltzmann surface area (MM/GBSA) method as implemented in the GROMACS tool [46]. In our study, the free binding energy (ΔG) was calculated by using van der Waals (ΔEvdW), electrostatic (ΔEelec), polar solvation (ΔGpolar) and nonpolar (ΔGnon-polar) energy contributions.

Biosensor-binding studies

To determine the difference between the binding affinity of MtDnaB-NTD for MtDnaG-CTD and those of MtDnaB-NTD for the MtDnaG-CTD mutants and other bacterial DnaG-CTDs, biosensor-based binding experiments were performed using an Autolab surface plasmon resonance (SPR) instrument at the Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi, India. The surface of SPR chip (a self-assembled monolayer of 11-mercaptoundecanoic acid on a gold surface; Autolab) was first activated with carbodiimide (EDC; 0.2 M) and N-hydroxysuccinimide (NHS; 0.05 M)/N-ethyl-N-(diethyl aminopropyl). MtDnaG-CTD, MtDnaG-CTD mutants and other bacterial DnaG-CTDs were immobilized (separately on different chips) onto the activated sensor surface at a concentration of 20 µg/ml in filtered (0.22-µm pore size) and degassed 10 mM sodium acetate buffer (pH 4.5). The chip had two channels: channel one was used for immobilization of ligand and channel two was used as a blank (the signals of the analyte with a ligand-free surface). After ligand immobilization, the surface was blocked with 100 mM ethanolamine at pH 8.5, followed by regeneration using 1 M NaCl. The running buffer constituents were the same as those recommended for HBS BIAcore running buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA and 0.05% P-20 surfactant). The association kinetics for DnaG-CTDs was monitored for 400 s, followed by monitoring of the dissociation kinetics for the next 300 s. MtDnaB-NTD samples of various concentrations were prepared in running buffer and injected at the rate of 20 µl/min across the sensor surface. The concentrations of MtDnaB-NTD used against MtDnaG-CTD were 1500, 1000, 750, 500, 250 and 125 nM, whereas, for the MtDnaG-CTD mutants and other bacterial DnaG-CTDs, the concentrations used were 2000, 1500, 1000, 750, 500 and 250 nM. Bovine serum albumin protein samples at various concentrations (2000, 1500, 1000, 750, 500 and 250 nM) were used as negative controls. Signal changes on the activated/blocked control panel were subtracted from the DnaG–DnaB-binding interactions using an inline reference signal, and the subtracted sensorgrams were analyzed. The surface was regenerated with a buffer consisting of two manually delivered pulses of 1 M NaCl. All data were recorded at 25°C. The data analysis was performed using the Autolab SPR kinetic evaluation software.

Results

The overall structure of MtDnaG-CTD

To obtain a more detailed view of the interaction between the helicase and primase of M. tuberculosis, we set out to determine a high-resolution crystal structure of the helicase-binding domain of MtDnaG (MtDnaG-CTD) and indeed did so to a resolution of 1.58 Å (Figure 2b). Molecular replacement failed to give any solution, indicative of the low homology and structural similarity with all of the previously determined DnaG-CTDs. Se-Met-labeled protein crystals were used to obtain anomalous data and experimental phases. The Se-Met-labeled protein crystal diffracted X-rays to 1.58 Å at BM14 of ESRF and belonged to the space group P21212 with cell dimensions of a = 48.2 Å, b = 137.5 Å, c = 36.5 Å (α = β = γ = 90°). The final model yielded an Rfactor of 18.1% and Rfree of 20.8%, with good electron density (Figure 2a). One complete MtDnaG-CTD (residues 481–637) and one helical hairpin cleaved from the MtDnaG-CTD (residues 569–637) were identified per asymmetric unit (Figure 2b) and these two protein chains were stabilized with a few van der Waals interactions (Figure 2d). The overall CTD structure of MtDnaG displayed two subdomains, the N-terminal globular region (GR) subdomain (residues 482–562) and C-terminal helical hairpin region (HHR) subdomain (residues 568–637), connected by a small loop (six residues) (Figure 2c). Inspection of the MtDnaG-CTD structure showed it to lack any β-sheet and to consist instead primarily of α-helices, specifically 10 of them with the first eight forming the GR and the last two forming the HHR (Figure 2c). The gel filtration chromatography profile of MtDnaG-CTD in a solution on a calibrated Superdex G75 16/60 column revealed a sharp elution peak at 70.3 ml, and an observed molecular mass of ∼38 kDa, indicating MtDnaG-CTD (∼19 kDa) to exist in a dimeric state in a solution (Figure 1). Electron density corresponding to the GR or N-terminal residues (residues 481–568) of MtDnaG-CTD was not visible in one monomer due to the cleavage of MtDnaG-CTD. MtDnaG-CTD cleaved into two fragments (GR and HHR), and the fragments were also identified using MALDI-TOF (Supplementary Figure S2). A total of 232 amino acid residues, one polyethylene glycol (PEG) molecule, one acetate molecule and 320 water molecules were determined to be present per asymmetric unit. The Ramachandran analysis showed 99.1% of the residues in the favored region, 0.5% in the generously allowed region and 0.4% in the disallowed region.

MtDnaG-CTD is a dimer in solution.

Figure 1.
MtDnaG-CTD is a dimer in solution.

(a) Size-exclusion chromatograph of the C-terminal domain of primase (MtDnaG-CTD) passed through a HiLoad 16/60 Superdex 75 column. Ten-milliliter fractions were collected. The elution volume (70.3 ml) and the elution pattern of the protein are displayed. (b) The molecular mass of the eluted DnaG-CTD was deduced from the standard plot to be ∼35 kDa, corresponding to the dimeric state of the protein. (c) SDS–PAGE showing fractions purified by gel filtration. The proteins were separated on SDS–12% PAGE and stained with Coomassie brilliant blue. Lane M shows the molecular markers; lane P shows the gel filtration fraction.

Figure 1.
MtDnaG-CTD is a dimer in solution.

(a) Size-exclusion chromatograph of the C-terminal domain of primase (MtDnaG-CTD) passed through a HiLoad 16/60 Superdex 75 column. Ten-milliliter fractions were collected. The elution volume (70.3 ml) and the elution pattern of the protein are displayed. (b) The molecular mass of the eluted DnaG-CTD was deduced from the standard plot to be ∼35 kDa, corresponding to the dimeric state of the protein. (c) SDS–PAGE showing fractions purified by gel filtration. The proteins were separated on SDS–12% PAGE and stained with Coomassie brilliant blue. Lane M shows the molecular markers; lane P shows the gel filtration fraction.

Crystal structure of MtDnaG-CTD.

Figure 2.
Crystal structure of MtDnaG-CTD.

(a) A portion of the molecule shown with a 2Fo − Fc electron density map contoured at 1σ within 1.5 Å of selected atoms. (b) Cartoon diagram showing the crystal structure of the MtDnaG primase CTD. The overall structure was observed to have one globular region connected to the helical hairpin region by a flexible loop. Chain A is colored purple, and chain B is cyan. One complete MtDnaG-CTD (chain A) and one helical hairpin cleaved from this CTD (chain B) were identified per asymmetric unit. (c) Cylindrical representation of the MtDnaG-CTD crystal structure showing the arrangement of the 10 helices connected by loops. Helices αH1–αH8 formed the globular region and helices αH9–αH10 formed the helical hairpin region. (d) Amino acid residues showed the hydrophobic interaction between the chain A and chain B in the crystal structure of MtDnaG-CTD.

Figure 2.
Crystal structure of MtDnaG-CTD.

(a) A portion of the molecule shown with a 2Fo − Fc electron density map contoured at 1σ within 1.5 Å of selected atoms. (b) Cartoon diagram showing the crystal structure of the MtDnaG primase CTD. The overall structure was observed to have one globular region connected to the helical hairpin region by a flexible loop. Chain A is colored purple, and chain B is cyan. One complete MtDnaG-CTD (chain A) and one helical hairpin cleaved from this CTD (chain B) were identified per asymmetric unit. (c) Cylindrical representation of the MtDnaG-CTD crystal structure showing the arrangement of the 10 helices connected by loops. Helices αH1–αH8 formed the globular region and helices αH9–αH10 formed the helical hairpin region. (d) Amino acid residues showed the hydrophobic interaction between the chain A and chain B in the crystal structure of MtDnaG-CTD.

The MtDnaG-CTD structure differs from other DnaG-CTD structures in the HHR

The 1.58-Å resolution of the current M. tuberculosis DnaG-CTD structure was higher than those of the other four available DnaG-CTD crystal structures, i.e. those determined for E. coli (resolution to 2.8 Å, PDB ID: 1T3W), B. stearothermophilus (2.9 Å, 2R6A), H. pylori (1.7 Å, 4EHS) and V. cholerae (2.4 Å, 4IM9) [15,16,53]. Structural alignments of MtDnaG-CTD with these structures were done, using LSQMAN [54], to evaluate the differences between them. We specifically aligned the GRs of these structures to show the diversity in the relative orientation of the HHR (Figure 3). The RMSDs between MtDnaG-CTD and EcDnaG-CTD, BstDnaGCTD, HpDnaG-CTD and VcDnaG-CTD were found to be 1.89 Å (45 Cα atoms), 1.85 Å (71 Cα atoms), 1.59 Å (62 Cα atoms) and 1.77 Å (43 Cα atoms), respectively. However, DnaG-CTDs have been found to share less than 10% sequence identity (Supplementary Figure S1). The total structural alignments of MtDnaG-CTD with EcDnaG-CTD, BstDnaGCTD, HpDnaG-CTD and VcDnaG-CTD using RAPIDO [55] yielded RMSDs of 12.58 Å (101 Cα atoms), 6.81 Å (131 Cα atoms), 5.24 Å (108 Cα atoms) and 4.28 Å (122 Cα atoms), respectively (Supplementary Table S2). Inspection of the superpositions of these structures suggested the GR of MtDnaG-CTD to be structurally more similar to that of HpDnaG-CTD than those of the other DnaG-CTDs. Overall, the arrangement of helices in MtDnaG-CTD was observed to be similar to that in VcDnaG-CTD (Supplementary Table S2). The number of α-helices present in each CTD structure was also found to differ: 10 helices in the MtDnaG-CTD structure, six each for HpDnaG-CTD and VcDnaG-CTD, and seven each for EcDnaG-CTD and BstDnaG-CTD (Supplementary Figure S4). These differences in the number of α-helices resulted, for the most part, in offsets between the boundaries of the helices in the structure-based alignment (Figure 3). Comparison of the MtDnaG-CTD structure with other DnaG-CTD structures showed a larger deviation between the entire structures than between the GRs. The HHRs from the different sources adopted different orientations relative to the GRs and showed much greater structural differences than did the GRs. Moreover, the B-factors of the HHRs and linkers were found to, in general, be greater than those of the GRs (Supplementary Table S3), indicating relatively high flexibility levels for the HHR and linker regions. Also, note the lower overall B-factor of the MtDnaG-CTD structure than of the other known CTD structures (Supplementary Table S3). Interestingly, despite all of the DnaG-CTD structures consisting of a GR as well as HHR, multiple sequence alignment of amino acid sequences (Supplementary Figure S1b) and structural alignment did not yield any significant homology.

Superpositions of the globular region of MtDnaG-CTD with the globular regions of other DnaG-CTDs provided insights into the differences in the shapes and orientations of HHRs.

Figure 3.
Superpositions of the globular region of MtDnaG-CTD with the globular regions of other DnaG-CTDs provided insights into the differences in the shapes and orientations of HHRs.

Superposition of the globular region of the MtDnaG-CTD (purple) crystal structure with the globular region of each of (a) EcDnaG-CTD (green), (b) VcDnaG-CTD (cyan), (c) BstDnaG-CTD (violet) and (d) HpDnaG-CTD (wheat). The helical hairpin regions (HHRs) of the different structures showed different orientations.

Figure 3.
Superpositions of the globular region of MtDnaG-CTD with the globular regions of other DnaG-CTDs provided insights into the differences in the shapes and orientations of HHRs.

Superposition of the globular region of the MtDnaG-CTD (purple) crystal structure with the globular region of each of (a) EcDnaG-CTD (green), (b) VcDnaG-CTD (cyan), (c) BstDnaG-CTD (violet) and (d) HpDnaG-CTD (wheat). The helical hairpin regions (HHRs) of the different structures showed different orientations.

MtDnaG-CTD interacts at the dimer–dimer interface of MtDnaB-NTD using mostly hydrophobic residues of the HHR

A previous investigation showed MtDnaB-NTD (residues 21–197, including the linker) to form a stable dimer in solution, and a truncated form of the protein (residues 21–134) to be monomeric, indicating the region spanning residues 135–197 to be required for dimer stability [28]. Therefore, we also generated these monomeric and dimeric MtDnaB-NTD constructs, using the same method as described earlier [28], for our studies. Specifically, biosensor-binding studies of each of these two constructs with MtDnaG-CTD were carried out using surface plasmon resonance by passing the respective MtDnaB-NTD construct on immobilized MtDnaG-CTD. The results of these two experiments showed MtDnaG-CTD to not interact with the monomeric form of MtDnaB-NTD (Supplementary Figure S3b) but to do so with the dimeric form (Supplementary Figure S3c).

The lack of a high-resolution structure of the DnaB–DnaG complex and the fragmented structural information for the individual proteins have impeded our attaining a detailed understanding of this crucial binary protein interaction. The only complex structure of the active hexameric BstDnaB (PDB: 2R6A) was determined as a trimer of dimer and three BstDnaG-CTDs were found at the dimer–dimer interface of BstDnaB [16]. Similarly, the crystal structure of the MtDnaB-NTD (PDB: 2R5U) has been reported to form a hexameric ring having two distinct interfaces: an extensive hydrobhobic interface, stabilizing a dimer (monomer–monomer) of MtDnaB-NTD, and other less extensive interface formed between the dimers, connecting three of them into a hexamer [28]. The dimer–dimer interface of MtDnaB-NTD was considered as a DnaG-binding site [28]. Here, we produced a model of the M. tuberculosis DnaB–DnaG complex by one molecule of MtDnaG-CTD interacted to two molecules of MtDnaB-NTD, involved at the dimer–dimer interface similar to the BstDnaB–DnaG-CTD complex structure [16]. Superimpositions of the GR of the MtDnaG-CTD crystal structure with GRs from the crystal structures of EcDnaG-CTD, VcDnaG-CTD, BstDnaG-CTD and HpDnaG-CTD (Figure 3) showed the orientation of the HHR to vary considerably between the different species and suggested the flexibility of the HHR. Inspection of the HHR in the unbound form of BstDnaG-CTD showed it to be oriented 65° away from its orientation in the helicase-bound form (Supplementary Figure S2), and this difference was attributed to the flexibility of the HHR. Therefore, the HHR of MtDnaG-CTD was rotated by 32° to model the MtDnaG-CTD/MtDnaB-NTD complex using the BstDnaB/DnaG-CTD (PDB: 2R6A) complex structure as a template (Supplementary Figure S5). A molecular dynamic simulation of the model was done for 25 ns and revealed the stability of the model after the first few nanoseconds of the simulation (Figure 4c). When producing our model of the MtDnaG-CTD/MtDnaB-NTD complex, we maximized the number of interactions between the HHR of MtDnaG-CTD and MtDnaB-NTD (Figure 4a). DnaG mutations in the part of the extreme C-terminal region, containing the HHR, were shown to be disruptive to the DnaB–DnaG complex [56], hence indicating this part of the C-terminal region to be crucial for the helicase–primase interaction. Our modeling highlighted the amino acid residues present in the helicase–primase complex interface that may participate in these crucial interactions and stabilize the complex (Figure 4a). The low homology between the DnaG-CTD sequences (Supplementary Figure S1a) together with the presence of different yet consistently hydrophobic amino acid residues at the M. tuberculosis (Figures 4a and 5b) and B. stearothermophilus [16] helicase–primase interface suggests the involvement of hydrophobic interactions in stabilizing the helicase–primase complex. According to the model, I605 and F615 from the primase of M. tuberculosis make extensive hydrophobic interactions with helicase, and these residues, in particular, may be playing a crucial role in stabilizing the helicase–primase complex (Figure 4b).

Model of the M. tuberculosis helicase–primase complex.

Figure 4.
Model of the M. tuberculosis helicase–primase complex.

(a) Model of the M. tuberculosis helicase–primase complex. Experimentally determined structures of MtDnaB-NTD (PDB: 2R5U) and MtDnaG-CTD (PDB: 5Z51), as well as that of the BstDnaB–BstDnaG-CTD complex (PDB: 2R6A), were used to generate the model of the complex. In the complex model, two molecules of DnaB-NTD interact with one molecule of DnaG-CTD. The model was further relaxed to eliminate bad contacts using molecular energy minimization with the help of the GROMACS molecular dynamic package. (b) The crucial residues of the helical hairpin showing hydrophobic interactions stabilizing the helicase–primase complex. (c) RMSD analysis of the trajectories of native and mutant primase models. Time-dependent Cα RMSDs for all residues of DnaB-NTD complexed with the native DnaG-CTD (black), I605A mutant DnaG-CTD (red) and F615A mutant DnaG-CTD (green) showed structural convergence from the initial time point. No effect of the mutations on the DnaG-CTD structure was observed, but a significant effect on the binding affinity of the helicase for the primase was found.

Figure 4.
Model of the M. tuberculosis helicase–primase complex.

(a) Model of the M. tuberculosis helicase–primase complex. Experimentally determined structures of MtDnaB-NTD (PDB: 2R5U) and MtDnaG-CTD (PDB: 5Z51), as well as that of the BstDnaB–BstDnaG-CTD complex (PDB: 2R6A), were used to generate the model of the complex. In the complex model, two molecules of DnaB-NTD interact with one molecule of DnaG-CTD. The model was further relaxed to eliminate bad contacts using molecular energy minimization with the help of the GROMACS molecular dynamic package. (b) The crucial residues of the helical hairpin showing hydrophobic interactions stabilizing the helicase–primase complex. (c) RMSD analysis of the trajectories of native and mutant primase models. Time-dependent Cα RMSDs for all residues of DnaB-NTD complexed with the native DnaG-CTD (black), I605A mutant DnaG-CTD (red) and F615A mutant DnaG-CTD (green) showed structural convergence from the initial time point. No effect of the mutations on the DnaG-CTD structure was observed, but a significant effect on the binding affinity of the helicase for the primase was found.

SPR sensorgram for measuring the binding affinity of the helicase for the primase.

Figure 5.
SPR sensorgram for measuring the binding affinity of the helicase for the primase.

Sensorgrams showing the binding pattern of MtDnaB-NTD at various concentrations (legends) with immobilized (a) native MtDnaG-CTD, (b) F615A mutant MtDnaG-CTD and (c) I605A mutant MtDnaG-CTD. The mutation of a single residue, Ile605 to Ala, on the helicase-binding surface resulted in an almost 10-fold decrease in the binding affinity.

Figure 5.
SPR sensorgram for measuring the binding affinity of the helicase for the primase.

Sensorgrams showing the binding pattern of MtDnaB-NTD at various concentrations (legends) with immobilized (a) native MtDnaG-CTD, (b) F615A mutant MtDnaG-CTD and (c) I605A mutant MtDnaG-CTD. The mutation of a single residue, Ile605 to Ala, on the helicase-binding surface resulted in an almost 10-fold decrease in the binding affinity.

Ile605 in the HHR is crucial for helicase–primase interaction in Mycobacterium tuberculosis

To elucidate the main drivers of the helicase–primase association and to identify hotspots, we turned to quantify individual contributions of potential key residues, guided by a structural analysis of our proposed M. tuberculosis DnaG-CTD/DnaB-NTD complex model. To accomplish this goal, we mutated a couple of crucial contact residues modeled to be present at the interface of the M. tuberculosis DnaG–DnaB complex. Inspection of our model (Figure 4a) indicated a large non-conserved hydrophobic residue, Ile605, to be the most prominent surface-accessible hydrophobic residue on the HHR of MtDnaG-CTD (Figure 4b). Inspection of our model indicated Ile605, of the loop connecting αH9 and αH10 of the HHR, to be stabilizing the complex. Ile605 presents at the crucial position, flexible loop within the HHR, through that the whole HHR gets hooked by this residue. Our analysis also indicated Phe615, another large hydrophobic residue of MtDnaG-CTD, to be present at the M. tuberculosis DnaG–DnaB interface (Figure 4b). This residue, being present at helix 10 of MtDnaG-CTD, was observed to be less accessible than MtDnaG-CTD Ile605 present at the connecting loop of the HHR. The stability levels of the helicase (MtDnaB-NTD) in complex with native MtDnaG-CTD, mutant MtDnaG-CTD F615A and mutant MtDnaG-CTD I605A were analyzed by carrying out molecular dynamic simulations (Figure 4c). Furthermore, the binding energy levels of all of the complex models were calculated using the MM/GBSA method of GROMACS (Supplementary Table S4). The binding energy values of helicase (MtDnaB-NTD) in complex with native MtDnaG-CTD, mutant MtDnaG-CTD F615A and mutant MtDnaG-CTD I605A were −100, −75.3 and −60 kj/mol, respectively (Supplementary Table S4). The binding affinities for the native and mutant MtDnaG-CTDs in complex with MtDnaB-NTD were also measured in vitro using SPR spectroscopy (Figure 5). Native and mutant MtDnaG-CTDs were immobilized on three different chips under identical conditions, and different concentrations of MtDnaB-NTD were passed as an analyte with HBS buffer over the immobilized proteins. The KD values of MtDnaB-NTD in complex with MtDnaG-CTD, MtDnaG-CTD F615A and MtDnaG-CTD I605A were measured to be 370, 444 and 2.8 µM, respectively (Figure 5). The SPR results indicated a 10-fold decrease in the binding affinity of MtDnaB-NTD with MtDnaG-CTD I605A compared with that of MtDnaB-NTD with native MtDnaG-CTD. Although the measured difference in binding affinity to the native (MtDnaG-CTD) and mutated (MtDnaG-CTD I605A) proteins was relatively large considering that only a single residue was mutated, there was still observed significant affinity between the helicase and the mutated (MtDnaG-CTD I605A) primase. In our current study, we noted the important role of the loop connecting the two helices of the HHR in stabilizing the helicase–primase complex. This major role of the loop within the HHR in the stability of the helicase–primase complex was indicated despite no common amino acid residues present at the same position of different DnaG-CTDs. Both in silico calculations and in vitro experiments (SPR) suggested a significant role of Ile605 of MtDnaG in the stabilization of the helicase–primase (MtDnaB/MtDnaG) complex in M. tuberculosis.

Mycobacterium DnaB-NTD interacts with DnaG-CTD of other organisms with low binding affinity

The helicase-binding domain of DnaG and primase-binding region of DnaB are the least conserved regions of their respective proteins. Despite the insignificant homology between the various DnaG-CTD sequences, the DnaG-CTD structures were found to be similar, especially in their all having globular and HHR regions. A functional complementation of EcDnaB with HpDnaB has been reported earlier [57], indicative of an interaction between HpDnaB and EcDnaG. To further test whether the cross-species helicase–primase interaction takes place even with such a low sequence similarity, we performed a study of the interaction of MtDnaB-NTD with other DnaG-CTDs. We studied the specificity in the interaction of DnaB with non-cognate DnaG by generating an in silico model of the complex and further tested the biophysical interactions of Mycobacterium DnaB-NTD with other DnaG-CTDs, i.e. from E. coli, H. pylori and V. cholerae. The steps used to in silico build and analyze the models of MtDnaB-NTD in complex with DnaG-CTDs from E. coli, H. pylori and V. cholerae (Figure 6) were similar to the strategy used in producing the model of the M. tuberculosis helicase–primase complex. The free energy values of the binding of native MtDnaB-NTD to EcDnaG-CTD, HpDnaG-CTD and VcDnaG-CTD were −13.7, −71.5 and −58.4 kJ/mol, respectively. The in silico studies suggested the MtDnaB-NTD/HpDnaG-CTD complex to be more stable than the MtDnaB-NTD/EcDnaG-CTD and MtDnaB-NTD/VcDnaG-CTD complexes. We also carried out biosensor-binding studies using SPR spectroscopy. Specifically, the binding affinities of native MtDnaB-NTD for EcDnaG-CTD, HpDnaG-CTD and VcDnaG-CTD were measured in vitro using SPR spectroscopy. The KD values for the complexes of MtDnaB-NTD with EcDnaG-CTD (Figure 7a) and HpDnaG-CTD (Figure 7b) were determined from these studies to be 7.4 and 3.4 µM, respectively, while MtDnaB-NTD formed an unstable association with VcDnaG-CTD (Figure 7c). However, the cross-species helicase–primase binding did not show the order as predicted by the in silico studies. But both in silico and in vitro cross-study on MtDnaB-NTD to DnaG-CTD of other bacteria showed the significant decrease in binding affinities compared with the binding affinity of native MtDnaG-CTD and MtDnaB-NTD interaction. Moreover, the decrease in the binding affinity of DnaG to DnaB might affect the activity of these proteins during the replication process.

In silico investigations of the cross-species helicase–primase complexes.

Figure 6.
In silico investigations of the cross-species helicase–primase complexes.

(ac) Experimentally determined structures of MtDnaB-NTD (PDB: 2R5U) and (a) EcDnaG-CTD (PDB: 1T3W), (b) HpDnaG-CTD (PDB: 4EHS) and (c) VcDnaG-CTD (PDB: 4IM9) were used to generate models of the helicase–primase complex. In each case, the crystal structure of the BstDnaB–BstDnaG-CTD (PDB: 2R6A) complex was used as a template. The model was further relaxed to eliminate bad contacts using molecular energy minimization with the help of the GROMACS molecular dynamic package. (d) RMSD analyses of the trajectories of the three models. Time-dependent Cα-RMSDs for all residues of DnaB-NTD complexed with EcDnaG-CTD (red), HpDnaG-CTD (black) and VcDnaG-CTD (green) showed structural convergence from the initial time point. There were no structural changes during the dynamic simulation, but the DnaB–DnaG-binding affinities for the cross-species complexes differed significantly from that for the native M. tuberculosis DnaB–DnaG complex.

Figure 6.
In silico investigations of the cross-species helicase–primase complexes.

(ac) Experimentally determined structures of MtDnaB-NTD (PDB: 2R5U) and (a) EcDnaG-CTD (PDB: 1T3W), (b) HpDnaG-CTD (PDB: 4EHS) and (c) VcDnaG-CTD (PDB: 4IM9) were used to generate models of the helicase–primase complex. In each case, the crystal structure of the BstDnaB–BstDnaG-CTD (PDB: 2R6A) complex was used as a template. The model was further relaxed to eliminate bad contacts using molecular energy minimization with the help of the GROMACS molecular dynamic package. (d) RMSD analyses of the trajectories of the three models. Time-dependent Cα-RMSDs for all residues of DnaB-NTD complexed with EcDnaG-CTD (red), HpDnaG-CTD (black) and VcDnaG-CTD (green) showed structural convergence from the initial time point. There were no structural changes during the dynamic simulation, but the DnaB–DnaG-binding affinities for the cross-species complexes differed significantly from that for the native M. tuberculosis DnaB–DnaG complex.

SPR sensorgrams for measuring the binding affinities of helicase with non-cognate primases.

Figure 7.
SPR sensorgrams for measuring the binding affinities of helicase with non-cognate primases.

Sensorgrams showing the binding patterns of MtDnaB-NTD at various concentrations (legends) with immobilized (a) E. coli DnaG-CTD, (b) H. pylori DnaG-CTD and (c) V. cholerea DnaG-CTD. The results indicated MtDnaB-NTD to weakly bind EcDnaG-CTD and HpDnaG-CTD, and unstably interact with VcDnaG-CTD.

Figure 7.
SPR sensorgrams for measuring the binding affinities of helicase with non-cognate primases.

Sensorgrams showing the binding patterns of MtDnaB-NTD at various concentrations (legends) with immobilized (a) E. coli DnaG-CTD, (b) H. pylori DnaG-CTD and (c) V. cholerea DnaG-CTD. The results indicated MtDnaB-NTD to weakly bind EcDnaG-CTD and HpDnaG-CTD, and unstably interact with VcDnaG-CTD.

Discussion

In this study, the crystal structure of the DnaB helicase-binding domain of DnaG primase (MtDnaG-CTD) of M. tuberculosis was determined to gain insights into the helicase–primase interaction, crucial for the survival of eubacteria. The complex between the DnaG primase and the DnaB helicase unwinds duplex DNA at the eubacterial replication fork and synthesizes the RNA primers required for initiation of DNA replication [58,59]. An alignment of DnaG-CTD sequences showed high diversity. Therefore, the MtDnaG-CTD crystal structure could not be solved using molecular replacement, and we instead used SAD phasing. Analysis of the determined structure highlighted the flexibility of the DnaG-CTD scaffold, which has been observed in other DnaG-CTDs, and supported the existence of hinge bending within the protein. The hinge point located in the linker between the GR and HHR apparently allows these two regions of the protein to become close together or far apart, resulting in an alternation between elongated and contracted shapes. Also, the flexibility of the helicase-binding domain has been shown to be due, in large part, to the identified hinge point and to be the main factor maximizing the interactions with DnaB helicase. A comparison of MtDnaG-CTD with other DnaG-CTD structures suggested some degree of conservation of the protein fold, in particular in the GR. Overall, the HHR appears to have the major role in the interaction with DnaB and shows a high degree of diversity in eubacteria.

We also constructed in silico a hypothetical model of the MtDnaB-NTD/MtDnaG-CTD complex, based on the BstDnaB/DnaG-CTD complex crystal structure [16], and found the hypothetical model to be consistent with previous mutagenesis studies [56]. In the model of the MtDnaB-NTD/MtDnaG-CTD complex, the HHR of MtDnaG-CTD covers the interface otherwise making dimer–dimer interactions in the hexameric form MtDnaB-NTD. Also, Ile605 and Phe615 are large non-conserved hydrophobic residues present at the HHR of MtDnaG and, according to our model, interact with MtDnaB (Figure 4a). Our analysis also indicated Ile605, a hydrophobic residue present on the loop of the HHR of MtDnaG-CTD, to interact with Val82 and Ala85 of MtDnaB-NTD and hence to be crucial for the formation of the complex. Our in silico study of the MtDnaB/MtDnaG complex also indicated a decrease in binding energy upon mutation of MtDnaG-CTD Ile605 to alanine as well as upon the mutation of its Phe615 to alanine (Supplementary Table S4). To provide further evidence for the results of these binding affinity calculations, we also carried out in vitro studies involving expressing both mutants of MtDnaG-CTD and using SPR spectroscopy to measure their affinities for DnaB-NTD. In this biosensor-binding study, the affinity of the Ile605Ala MTDnaG-CTD mutant for MtDnaB-NTD was shown to be 10 times weaker than the affinity of the native MtDnaG-CTD for MtDnaB-NTD. These findings helped us validate the model and identify Ile605 as one of the key MtDnaG residues stabilizing the association of MtDnaG with MtDnaB.

Analysis of our current structure, biosensor results and the results of the previous studies of DnaB–DnaG complexes [15,16] led us to conclude that the loop connecting the two helices of the HHR is apparently largely responsible for the stability of this complex. The sequence and structural analysis indicated the non-conserved residue Ile605, present at the helical hairpin loop of DnaG, to be specific to M. tuberculosis and to be a key residue for the interaction between DnaB and DnaG (Figure 4). The earlier study in H. pylori, residue Phe537, of the helical hairpin loop of DnaG-CTD has been shown to play a key role in the stability of the DnaB–DnaG complex [15]. The observation of amino acid residues present at the helical hairpin loop of DnaG-CTDs suggested the presence of non-conserved hydrophobic residues that play a significant role in the stability of the DnaB–DnaG complex. The flexibility of the loop apparently allows for maximizing the access of its hydrophobic residues for contacts with DnaB-NTD.

The binding affinity of MtDnaB for MtDnaG was measured to be in the nanomolar range while the binding affinities of MtDnaB with other DnaGs from different species were measured in the micromolar range (Figures 5 and 7). Such a relatively low level of heterologous binding affinity might be expected to result in relatively poor helicase and primase activities in the case of complementarity with other organisms. A suboptimal activity level of any DNA replication initiation protein would, in turn, be expected to slow down replication fork progression [2]. Though the helicase of H. pylori showed functional complementarity with E. coli [57], such complementarity of this helicase seems to be specific to just a few organisms. Therefore, MtDnaB may show complementarity with E. coli and H. pylori but not in V. cholerae. The pattern of interactions between DnaG and DnaB seems to vary from one organism to the next, and therefore, any change in the affinity between these proteins may affect not only the initiation of replication but also overall DNA replication.

Based on the crystal structure and our model, we can suggest a possible mechanism to explain the regulation of DnaB–DnaG activity at the replication fork. Bacterial primases primarily adopt a compact state but can transition to an extended conformation [60]. In the unbound form or free form of DnaG, the HHR may fold back to interfere with the RNA polymerase domain or the Zn-binding domain. According to the proposed mechanism, the moment when DnaG-CTD comes into contact to helicase, the GR side of DnaG-CTD might interact to the one molecule of MtDnaB-NTD and this binding might enforce for changing the orientation of HHR from the unbound state of DnaG. The HHR would bend in such a way as to properly bind to the second molecule of DnaB-NTD with comparatively stronger than the first one, and the bending of the HHR away from the RNA polymerase domain or Zn-binding domain may lead to the activation of primase by coming into contact with DnaB. Because DnaG cannot effectively function as an independent molecule, i.e. unbound from DnaB, this mechanism would also provide a means to prevent unwanted priming at non-replicative sites in the cell. Future studies to further address this model in the context of the replisome will highlight how interactions between DnaG and the rest of the replisome synergize to regulate replication of the lagging strand.

In conclusion, the structural and biophysical data provided by our work have shed light on the structural assembly and mode of binding of DnaG-CTD with DnaB-NTD in M. tuberculosis and unveiled mechanistic details and selectivity determinants for other eubacteria. The non-conserved interacting domains of DnaG–DnaB complex expand the study on the aspects of the species-specific evolution of the replication system.

Abbreviations

     
  • Bst

    Bacillus stearothermophilus

  •  
  • DnaB

    N-terminal domain of helicase

  •  
  • DnaB-NTD

    DnaB N-terminal domain

  •  
  • DnaG

    C-terminal domain of primase

  •  
  • DnaG-CTD

    DnaG C-terminal domain

  •  
  • Ec

    Escherichia coli

  •  
  • GR

    globular region

  •  
  • HHR

    helical hairpin region

  •  
  • Hp

    Helicobacter pylori

  •  
  • IPTG

    isopropyl-beta-d-thiogalactopyranoside

  •  
  • LB

    Luria-Bertani

  •  
  • MM/GBSA

    molecular mechanics Poisson–Boltzmann surface area

  •  
  • Mt

    Mycobacterium tuberculosis

  •  
  • MtDnaG-CTD

    DnaG from Mycobacterium tuberculosis

  •  
  • PCR

    polymerase chain reaction

  •  
  • PEG

    polyethylene glycol

  •  
  • PMSF

    phenylmethyl sulfonyl fluoride

  •  
  • RMSD

    root mean square deviation

  •  
  • RMSD

    root-mean-square deviation

  •  
  • Se-Met

    Selenomethionine

  •  
  • SPR

    surface plasmon resonance

  •  
  • ssDNA

    single-stranded DNA

  •  
  • Vc

    Vibrio cholerae

Author Contribution

S.G. and D.P.S. conceived the idea and designed the experiments. D.P.S. solved the MtDnaG-CTD structure. S.G., D.P.S. and R.V. analyzed the structures and constructed the models. D.P.S. generated S.D.M. and performed all of the biosensor studies. R.V. performed the MD simulations of all of the models and SAAR performed cloning. D.P.S. wrote the manuscript taking input from S.G., and S.G. reviewed the manuscript.

Acknowledgments

S.G. acknowledges DBT, DST-PURSE and UGC RNW, University Grant Commission (SAP). D.P.S. acknowledges UGC for the fellowship and R.V. acknowledges DST-SERB for the fellowship. We thank the Advanced Instrumentation Research Facility (AIRF) and JNU for providing the SPR facility. We also thank the staffs of BM14, ESRF for helping us with the collection of high-resolution X-ray data.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Kornberg
,
A.
and
Baker
,
T.A.
(
1980
)
DNA Replication
,
W.H. Freeman
,
San Francisco
2
Mott
,
M.L.
and
Berger
,
J.M.
(
2007
)
DNA replication initiation: mechanisms and regulation in bacteria
.
Nat. Rev. Microbiol.
5
,
343
354
3
Fang
,
L.
,
Davey
,
M.J.
and
O'Donnell
,
M.
(
1999
)
Replisome assembly at oriC, the replication origin of E. coli, reveals an explanation for initiation sites outside an origin
.
Mol. Cell
4
,
541
553
4
LeBowitz
,
J.
and
McMacken
,
R.
(
1986
)
The Escherichia coli dnaB replication protein is a DNA helicase
.
J. Biol. Chem.
261
,
4738
4748
PMID:
[PubMed]
5
Hacker
,
K.J.
and
Johnson
,
K.A.
(
1997
)
A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding
.
Biochemistry
36
,
14080
14087
6
O'Donnell
,
M.E.
and
Li
,
H.
(
2018
)
The ring-shaped hexameric helicases that function at DNA replication forks
.
Nat. Struct. Mol. Biol.
25
,
120
130
7
Corn
,
J.E.
and
Berger
,
J.M.
(
2006
)
Regulation of bacterial priming and daughter strand synthesis through helicase-primase interactions
.
Nucleic Acids Res.
34
,
4082
4088
8
Van Eijk
,
E.
,
Paschalis
,
V.
,
Green
,
M.
,
Friggen
,
A.H.
,
Larson
,
M.A.
,
Spriggs
,
K.
et al. 
(
2016
)
Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile
.
Open Biol.
6
,
160272
9
Bird
,
L.E.
,
Pan
,
H.
,
Soultanas
,
P.
and
Wigley
,
D.B.
(
2000
)
Mapping protein–protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus
.
Biochemistry
39
,
171
182
10
Tougu
,
K.
and
Marians
,
K.J.
(
1996
)
The interaction between helicase and primase sets the replication fork clock
.
J. Biol. Chem.
271
,
21398
21405
11
Tougu
,
K.
,
Peng
,
H.
and
Marians
,
K.J.
(
1994
)
Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork
.
J. Biol. Chem.
269
,
4675
4682
PMID:
[PubMed]
12
Davey
,
M.J.
,
Jeruzalmi
,
D.
,
Kuriyan
,
J.
and
O'Donnell
,
M.
(
2002
)
Motors and switches: AAA+ machines within the replisome
.
Nat. Rev. Mol. Cell Biol.
3
,
826
835
13
Mitkova
,
A.V.
,
Khopde
,
S.M.
and
Biswas
,
S.B.
(
2003
)
Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis
.
J. Biol. Chem.
278
,
52253
52261
14
Kashav
,
T.
,
Nitharwal
,
R.
,
Abdulrehman
,
S.A.
,
Gabdoulkhakov
,
A.
,
Saenger
,
W.
,
Dhar
,
S.K.
et al. 
(
2009
)
Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori
.
PLoS ONE
4
,
e7515
15
Rehman
,
S.A.A.
,
Verma
,
V.
,
Mazumder
,
M.
,
Dhar
,
S.K.
and
Gourinath
,
S.
(
2013
)
Crystal structure and mode of helicase binding of the C-terminal domain of primase from Helicobacter pylori
.
J. Bacteriol.
195
,
2826
2838
16
Bailey
,
S.
,
Eliason
,
W.K.
and
Steitz
,
T.A.
(
2007
)
Structure of hexameric DnaB helicase and its complex with a domain of DnaG primase
.
Science
318
,
459
463
17
Pai
,
M.
,
Behr
,
M.A.
,
Dowdy
,
D.
,
Dheda
,
K.
,
Divangahi
,
M.
,
Boehme
,
C.C.
et al. 
(
2016
)
Tuberculosis
.
Nature Rev. Dis. Primers
2
,
16076
18
Grosset
,
J.
(
2003
)
Mycobacterium tuberculosis in the extracellular compartment: an underestimated adversary
.
Antimicrob. Agents Chemother.
47
,
833
836
19
Wayne
,
L.G.
and
Hayes
,
L.G.
(
1996
)
An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence
.
Infect. Immun.
64
,
2062
2069
PMID:
[PubMed]
20
Boshoff
,
H.I.
,
Reed
,
M.B.
,
Barry
,
C.E.
and
Mizrahi
,
V.
(
2003
)
Dnae2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis
.
Cell
113
,
183
193
21
Katayama
,
T.
,
Ozaki
,
S.
,
Keyamura
,
K.
and
Fujimitsu
,
K.
(
2010
)
Regulation of the replication cycle: conserved and diverse regulatory systems for DnaA and oriC
.
Nat. Rev. Microbiol.
8
,
163
170
22
Bell
,
S.P.
and
Kaguni
,
J.M.
(
2013
)
Helicase loading at chromosomal origins of replication
.
Cold Spring Harb. Perspect. Biol.
5
,
a010124
23
Velten
,
M.
,
McGovern
,
S.
,
Marsin
,
S.
,
Ehrlich
,
S.D.
,
Noirot
,
P.
and
Polard
,
P.
(
2003
)
A two-protein strategy for the functional loading of a cellular replicative DNA helicase
.
Mol. Cell
11
,
1009
1020
24
Brézellec
,
P.
,
Vallet-Gely
,
I.
,
Possoz
,
C.
,
Quevillon-Cheruel
,
S.
and
Ferat
,
J.-L.
(
2016
)
Dcia is an ancestral replicative helicase operator essential for bacterial replication initiation
.
Nat. Commun.
7
,
13271
25
Mann
,
K.M.
,
Huang
,
D.L.
,
Hooppaw
,
A.J.
,
Logsdon
,
M.M.
,
Richardson
,
K.
,
Lee
,
H.J.
et al. 
(
2017
)
Rv0004 is a new essential member of the mycobacterial DNA replication machinery
.
PLoS Genet.
13
,
e1007115
26
Zhang
,
H.
,
Zhang
,
Z.
,
Yang
,
J.
and
He
,
Z.G.
(
2014
)
Functional characterization of DnaB helicase and its modulation by single-stranded DNA binding protein in Mycobacterium tuberculosis
.
FEBS J.
281
,
1256
1266
27
Yamamoto
,
K.
,
Low
,
B.
,
Rutherford
,
S.A.
,
Rajagopalan
,
M.
and
Madiraju
,
M.V.
(
2001
)
The Mycobacterium avium-intracellulare complex dnaB locus and protein intein splicing
.
Biochem. Biophys. Res. Commun.
280
,
898
903
28
Biswas
,
T.
and
Tsodikov
,
O.V.
(
2008
)
Hexameric ring structure of the N-terminal domain of Mycobacterium tuberculosis DnaB helicase
.
FEBS J.
275
,
3064
3071
29
Syson
,
K.
,
Thirlway
,
J.
,
Hounslow
,
A.M.
,
Soultanas
,
P.
and
Waltho
,
J.P.
(
2005
)
Solution structure of the helicase-interaction domain of the primase DnaG: a model for helicase activation
.
Structure
13
,
609
616
30
Otwinowski
,
Z.
and
Minor
,
W.
(
1997
)
[20] Processing of X-ray diffraction data collected in oscillation mode
.
Methods Enzymol.
276
,
307
326
31
Collaborative Computational Project, Number 4
. (
1994
)
The CCP4 suite: programs for protein crystallography
.
Acta Crystallogr. D Biol. Crystallogr.
50
,
760
763
32
Winn
,
M.D.
,
Ballard
,
C.C.
,
Cowtan
,
K.D.
,
Dodson
,
E.J.
,
Emsley
,
P.
,
Evans
,
P.R.
et al. 
(
2011
)
Overview of the CCP4 suite and current developments
.
Acta Crystallogr. D Biol. Crystallogr.
67
,
235
242
33
Panjikar
,
S.
,
Parthasarathy
,
V.
,
Lamzin
,
V.S.
,
Weiss
,
M.S.
and
Tucker
,
P.A.
(
2005
)
Auto-Rickshaw: an automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment
.
Acta Crystallogr. D Biol. Crystallogr.
61
,
449
457
34
Sheldrick
,
G.M.
(
2010
)
Experimental phasing with SHELXC/D/E: combining chain tracing with density modification
.
Acta Crystallogr. D Biol. Crystallogr.
66
,
479
485
35
Schneider
,
T.R.
and
Sheldrick
,
G.M.
(
2002
)
Substructure solution with SHELXD
.
Acta Crystallogr. D Biol. Crystallogr.
58
,
1772
1779
36
Hao
,
Q.
(
2004
)
ABS: a program to determine absolute configuration and evaluate anomalous scatterer substructure
.
J. Appl. Crystallogr.
37
,
498
499
37
Sheldrick
,
G.M.
(
2002
)
Macromolecular phasing with SHELXE
.
Z. Kristallogr. Cryst. Mater.
217
,
644
650
38
Terwilliger
,
T.C.
(
2000
)
Maximum-likelihood density modification
.
Acta Crystallogr. D Biol. Crystallogr.
56
,
965
972
39
Perrakis
,
A.
,
Morris
,
R.
and
Lamzin
,
V.S.
(
1999
)
Automated protein model building combined with iterative structure refinement
.
Nat. Struct. Mol. Biol.
6
,
458
40
Emsley
,
P.
and
Cowtan
,
K.
(
2004
)
Coot: model-building tools for molecular graphics
.
Acta Crystallogr. D Biol. Crystallogr.
60
,
2126
2132
41
Murshudov
,
G.N.
,
Vagin
,
A.A.
and
Dodson
,
E.J.
(
1997
)
Refinement of macromolecular structures by the maximum-likelihood method
.
Acta Crystallogr. D Biol. Crystallogr.
53
,
240
255
42
Emsley
,
P.
,
Lohkamp
,
B.
,
Scott
,
W.G.
and
Cowtan
,
K.
(
2010
)
Features and development of coot
.
Acta Crystallogr. D Biol. Crystallogr.
66
,
486
501
43
Murshudov
,
G.N.
,
Skubák
,
P.
,
Lebedev
,
A.A.
,
Pannu
,
N.S.
,
Steiner
,
R.A.
,
Nicholls
,
R.A.
et al. 
(
2011
)
REFMAC5 for the refinement of macromolecular crystal structures
.
Acta Crystallogr. D Biol. Crystallogr.
67
,
355
367
44
Laskowski
,
R.A.
,
MacArthur
,
M.W.
,
Moss
,
D.S.
and
Thornton
,
J.M.
(
1993
)
PROCHECK: a program to check the stereochemical quality of protein structures
.
J. Appl. Crystallogr.
26
,
283
291
45
DeLano
,
W.L.
(
2002
)
The PyMOL Molecular Graphics System
. http://pymol.org
46
Kumari
,
R.
,
Kumar
,
R.
,
Consortium
,
O.S.D.D.
and
Lynn
,
A.
(
2014
)
G_mmpbsa — a GROMACS tool for high-throughput MM-PBSA calculations
.
J. Chem. Inform. Model.
54
,
1951
1962
47
Abraham
,
M.J.
,
Murtola
,
T.
,
Schulz
,
R.
,
Páll
,
S.
,
Smith
,
J.C.
,
Hess
,
B.
et al. 
(
2015
)
GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers
.
SoftwareX
1–2
,
19
25
48
Wu
,
Y.
,
Tepper
,
H.L.
and
Voth
,
G.A.
(
2006
)
Flexible simple point-charge water model with improved liquid-state properties
.
J. Chem. Phys.
124
,
024503
49
Berendsen
,
H.J.
,
Jv
,
P.
,
van Gunsteren
,
W.F.
,
DiNola
,
A.
and
Haak
,
J.
(
1984
)
Molecular dynamics with coupling to an external bath
.
J. Chem. Phys.
81
,
3684
3690
50
Parrinello
,
M.
and
Rahman
,
A.
(
1981
)
Polymorphic transitions in single crystals: a new molecular dynamics method
.
J. Appl. Phys.
52
,
7182
7190
51
Darden
,
T.
,
York
,
D.
and
Pedersen
,
L.
(
1993
)
Particle mesh Ewald: an N⋅log (N) method for Ewald sums in large systems
.
J. Chem. Phys.
98
,
10089
10092
52
Hess
,
B.
,
Bekker
,
H.
,
Berendsen
,
H.J.
and
Fraaije
,
J.G.
(
1997
)
LINCS: a linear constraint solver for molecular simulations
.
J. Comput. Chem.
18
,
1463
1472
53
Oakley
,
A.J.
,
Loscha
,
K.V.
,
Schaeffer
,
P.M.
,
Liepinsh
,
E.
,
Pintacuda
,
G.
,
Wilce
,
M.C.
et al. 
(
2005
)
Crystal and solution structures of the helicase-binding domain of Escherichia coli primase: o
.
J. Biol. Chem.
280
,
11495
11504
54
Armougom
,
F.
,
Moretti
,
S.
,
Keduas
,
V.
and
Notredame
,
C.
(
2006
)
The iRMSD: a local measure of sequence alignment accuracy using structural information
.
Bioinformatics
22
,
e35
ee9
55
Hasegawa
,
H.
and
Holm
,
L.
(
2009
)
Advances and pitfalls of protein structural alignment
.
Curr. Opin. Struct. Biol.
19
,
341
348
56
Tougu
,
K.
and
Marians
,
K.J.
(
1996
)
The extreme C terminus of primase is required for interaction with DnaB at the replication fork
.
J. Biol. Chem.
271
,
21391
21397
57
Soni
,
R.K.
,
Mehra
,
P.
,
Mukhopadhyay
,
G.
and
Dhar
,
S.K.
(
2005
)
Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo
.
Biochem. J.
389
,
541
548
58
Ha
,
T.
,
Rasnik
,
I.
,
Cheng
,
W.
,
Babcock
,
H.P.
,
Gauss
,
G.H.
,
Lohman
,
T.M.
et al. 
(
2002
)
Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase
.
Nature
419
,
638
59
Lu
,
Y.-B.
,
Ratnakar
,
P.V.
,
Mohanty
,
B.K.
and
Bastia
,
D.
(
1996
)
Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA
.
Proc. Natl Acad. Sci. U.S.A.
93
,
12902
12907
60
Corn
,
J.E.
,
Pease
,
P.J.
,
Hura
,
G.L.
and
Berger
,
J.M.
(
2005
)
Crosstalk between primase subunits can act to regulate primer synthesis in trans
.
Mol. Cell
20
,
391
401