DNA backbone interactions impact the sequence specificity of DNA sulfur-binding domains: revelations from structural analyses

Abstract

The sulfur atom of phosphorothioated DNA (PT-DNA) is coordinated by a surface cavity in the conserved sulfur-binding domain (SBD) of type IV restriction enzymes. However, some SBDs cannot recognize the sulfur atom in some sequence contexts. To illustrate the structural determinants for sequence specificity, we resolved the structure of SBD_Spr, from endonuclease SprMcrA, in complex with DNA of G_PSGCC, G_PSATC and G_PSAAC contexts. Structural and computational analyses explained why it binds the above PT-DNAs with an affinity in a decreasing order. The structural analysis of SBD_Spr–G_PSGCC and SBD_Sco–G_PSGCC, the latter only recognizes DNA of G_PSGCC, revealed that a positively charged loop above the sulfur-coordination cavity electrostatically interacts with the neighboring DNA phosphate linkage. The structural analysis indicated that the DNA–protein hydrogen bonding pattern and weak non-bonded interaction played important roles in sequence specificity of SBD protein. Exchanges of the positively-charged amino acid residues with the negatively-charged residues in the loop would enable SBD_Sco to extend recognization for more PT-DNA sequences, implying that type IV endonucleases can be engineered to recognize PT-DNA in novel target sequences.

INTRODUCTION

Bacterial DNA phosphorothioate (PT) modification involves the replacement of the R_P non-bridging oxygen of a given phosphodiester bond with sulfur by Dnd proteins (1,2). Natural PT modifications dynamically occur to both DNA strands at the consensus sequences of G_PSGCC (_PS denotes PT link) in Streptomyces lividans 66, G_PSAAC/ G_PSTTC in Escherichia coli B7A and Salmonella enterica 87, and G_PSATC in Bermanella marisrubri RED65, or to a single strand at C_PSCA sites in Vibrio cyclitrophicus FF75 (3,4). dnd gene clusters governing PT modification are present in more than 1,300 bacterial and archaeal species (5,6). PT modification has been implicated in conferring resistance to oxidation to the host bacteria (7,8), influencing the global transcriptional response (9), and participating in restriction-modification systems in bacteria (10).

DNA modifications, primarily base methylation, participate in DNA replication and gene regulation through interactions with different nucleic acid binding proteins, also known as ‘readers’ (11), which transmit methylation information to other systems. For example, 5mCpG, the major eukaryotic methylated dinucleotide, is recognized by the methyl-GpG-binding (MBD) domain and the SET and RING finger-associated (SRA) domain, the prevalent 5mC reader in three life kingdoms (12,13). The SRA domain is often fused to other domain(s) that function in versatile cellular processes related to 5mC metabolism, or fused with a nuclease motif to cleave DNA in a modification-dependent way (14–18). Therefore, studies of the recognition mechanism of DNA modification by these readers are important to understand the flow of epigenetic information.

We recently identified a new type of reader in the Streptomyces coelicolor type IV restriction enzyme ScoMcrA, which specifically recognizes and cleaves PT-modified DNA (19), and this reader is a sulfur-binding domain (SBD) (20,21). PT-dependence is dictated by SBDs, whose carboxyl termini almost exclusively contain HNH nuclease motifs (21).

The complex structure of SBD_Sco bound to the PT-DNA sequence 5′-CCG_PSGCCGG-3′ was determined [Protein Data Bank (PDB) accession number: 5ZMO] (20). Discrimination of sulfur from oxygen by the SBD was achieved by concurrent interactions with the sulfur atom, the non-bridging oxygen of neighboring phosphates, as well as with the base pairs surrounding the PT linkage, in a manner generally similar to the recognition of 5mC or 5hmC by SRA. SBD_Sco only contacted the PT linkage on one DNA strand even though both strands were phosphorothioated. Three phosphates in the vicinity of the central PT link were wrapped in a cleft edged by two positively-charged patches on the surface of SBD_Sco, where the R_P sulfur was firmly coordinated by a hydrophobic concave pointing to the bottom of this cleft (20). SBD_Sco only binds to PT-DNA in the sequence G_PSGCC and not to the other four natural PT modification sequences, in sharp contrast to that the SRA domain has high flexibility in target sequence selection (22). In the complex structure of SBD_Sco–G_PSGCC, four residues on a ‘base-contacting’ loop formed seven hydrogen bonds with the three base pairs across the PT link. Single mutation of individual residues lowered the binding affinity to G_PSGCC by varying amounts, ranging from 20% to 80% (20). By contrast, the SBD of the PT-dependent restriction endonuclease (REase) SprMcrA from Streptomyces pristinaespiralis has a relatively relaxed sequence specificity, targeting G_PSGCC, G_PSAAC and G_PSATC, but not G_PSTTC or C_PSCA (21).

To understand the reasons underlying the differences in sequence specificity among SBDs, we crystalized SBD_Spr with G_PSGCC, G_PSAAC and G_PSATC. Comparative structural analysis revealed that a surface patch on two SBDs possesses a reverse charge, which exerts repelling and attracting strength on DNA by SBD_Sco and SBD_Spr, respectively. Mutation of E156R/D157R in this patch from SBD_Sco conferred the mutant domain with the ability to bind G_PSAAC and G_PSATC. Additionally, we provide evidence for why both SBDs showed a higher affinity for G_PSGCC than for the other DNA sequences. This study reports that variation in DNA binding affinity constitutes a key determinant of the sequence specificity for SBDs and provides new insights into approaches for engineering the specificity of modification-dependent REases by altering their contacts with DNA phosphates other than the nucleotide bases.

MATERIALS AND METHODS

Construction of protein expression vector and site-directed mutagenesis

DNA fragments encoding wild-type SBD_Spr and SBD_Sco were cloned into the pET28a vector (Novagen), with N-terminal 6xHis tags. His-tagged SBD_Spr and SBD_Sco mutant variants were constructed by the whole-plasmid PCR and DpnI digestion method (23). The Escherichia coli strain DH10b was used as a transformation host. The mutations were confirmed by DNA sequencing of the entire gene. Primers used for plasmid construction were listed in Supplementary Table S1.

Preparation and purification of stereospecific PT-DNA

The PT-DNA oligonucleotides were chemically synthesized and PAGE-purified. The concentration of oligonucleotides was determined by spectrophotometric measurement on a NanoDrop 2000 spectrophotometer (Thermo), and double-stranded DNA was prepared by mixing equimolar concentrations of complementary oligonucleotides, followed by heating to 95°C for 2 min and gradual cooling. The R_P and S_P stereoisomers of double-stranded PT-DNA were separated by anion exchange HPLC with a DNAPac PA-100 analytical column (Thermo) on an Agilent 1260 Infinity Series system at a flow rate of 1 ml/min with the following parameters (column at room temperature; solvent A, 10 mM Tris–HCl, pH 8.0; solvent B, 10 mM Tris–HCl, pH 8.0, 1 M NaCl; gradient, 10% B to 70% B over 40 min; detection by UV absorbance at 260 nm). The eluent was desalted with a Copure C18 column (Biocomma), dried on an RVC 2–25 rotational vacuum concentrator (Martin Christ), and dissolved with distilled deionized water.

Protein expression and purification

Proteins were expressed in the Escherichia coli strain BL21(DE3) at 16°C; a 10-ml culture grown overnight from a single colony was inoculated into 1 l of Luria Broth medium supplied with 50 ug/ml kanamycin. The culture was incubated at 37°C to an OD₆₀₀ of 0.6–0.8 and induced by the addition of 0.2 mM isopropyl-d-1-thiogalactopyranoside (IPTG) for another 20 h at 16°C. The cells were harvested and resuspended in 20 ml binding buffer (20 mM MES, pH 6.8, 20 mM imidazole, and 300 mM NaCl) and lysed by sonication in an ice bath. After centrifugation at 16 000 g for 60 min at 4°C, the supernatant was applied to 2 ml Ni-NTA column (GE Healthcare) pre-equilibrated with binding buffer. The Ni-NTA column was eluted with 10 ml of elution buffer (20 mM MES, pH 6.8, 300 mM imidazole and 300 mM NaCl) after washing. The His₆-tagged protein products were purified with a HiTrap Heparin HP affinity chromatography column (GE Healthcare), and a Superdex200 10/300 GL gel filtration chromatography column (GE Healthcare) equilibrated with 10 mM Tris–HCl (pH 8.0), 100 mM NaCl and 1 mM DTT, using an AKTA FPLC system (GE Healthcare). The peak fractions were combined and concentrated to 10 mg/ml. Purified proteins were visualized by Coomassie-stained 15% SDS-PAGE analysis, and protein concentration was determined using a Bradford Protein Assay Kit (Bio-Rad).

Crystallization, data collection and structure determination

Crystals for SBD_Spr in complex with the R_P form of the 8-bp hemi-PT DNA oligonucleotide 5′-GGCG_PSGCCC-3′ were grown at 14°C using the sitting-drop vapor-diffusion method in 48-well plates (24). Typically, 1 μl of reservoir solution was mixed with 1 μl of protein–DNA solution and equilibrated against 80 μl of reservoir solution. After optimization and macroseeding efforts, diffracting crystals of SBD_Spr–G_PSGCC were obtained from a buffer of 0.01 M magnesium acetate tetrahydrate, 0.05 M sodium cacodylate trihydrate pH 6.5, and 1.3 M lithium sulfate monohydrate. Crystal diffraction datasets at a resolution of 2.06 Å for the SBD_Spr–G_PSGCC complex were collected at the BL19U1 beamline at the National Center for Protein Science Shanghai and processed using HKL2000 (25). The crystal belonged to space group P6₁22, and contained three molecules of SBD_Spr in complex with three molecules of PT-DNA in each asymmetric unit. The crystal structure was determined by the molecular replacement method with the Phaser program (26), using the structure of SBD_Spr–G_PSAAC as the searching model. The structure of the SBD_Spr–G_PSGCC complex was refined and rebuilt using Coot (27) and Refmac (28).

The co-crystal of SBD_Spr with 8-bp oligos with G_PSAAC sequence was not successfully obtained. Crystals for SBD_Spr in complex with the R_P form of the 10-bp hemi-PT DNA oligonucleotide 5′-GGCG_PSAACGTG-3′ were grown and obtained at 14°C with the reservoir solution containing 0.1 M BIS-Tris pH 5.5, 0.15 M ammonium acetate, and 25% PEG 3350. The SBD_Spr–G_PSAAC complex crystals belonged to the P1 space group, with two molecules of SBD_Spr and two molecules of G_PSAAC–DNA; the structure of the complex was determined to 2.42 Å by the molecular replacement method with the phenix.rosetta_refine program (29), using the SBD domain of the ScoMcrA structure (PDB code: 5ZMO) as the searching model. The structure of the SBD_Spr-G_PSAAC complex was refined and rebuilt using Coot and Phenix.refine.

Crystals for SBD_Spr in complex with the R_P form of the 8-bp hemi-PT DNA oligonucleotide 5′-GATG_PSATCC-3′ were grown at 14°C with the reservoir solution containing 0.1 M Tris–HCl pH 8.5 and 4.5% PEG 8000. The SBD_Spr–G_PSATC complex crystals belonged to the C222₁ space group, with two molecules of SBD_Spr and two molecules of G_PSATC–DNA in the asymmetric unit; the structure of this complex was determined to 3.3 Å by the molecular replacement method with the Phaser program (30), using the structure of SBD_Spr–G_PSAAC as the searching model. The structure of the SBD_Spr–G_PSATC complex was refined and rebuilt using Coot, Refmac and Phenix.refine.

The data collection statistics and the refinement statistics for the SBD_spr–G_PSGCC, SBD_Spr–G_PSAAC and SBD_Spr–G_PSATC complexes are summarized in Supplementary Table S2.

Electrophoretic mobility shift assay (EMSA)

Each EMSA reaction contained 6 pmol DNA and protein at a concentration 4-fold higher than the DNA concentration (molar ratio) in 10μl binding buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl and 5% glycerol). After incubation at room temperature for 5 minutes, the reaction mixtures were loaded onto 12% non-denaturing polyacrylamide gels (acrylamide:bisacrylamide ratio of 79:1, w/w) and electrophoresed in 0.5× TBE buffer at 15 mA for 30 min. Ten bp-oligonucleotides used for EMSA assay were listed in Supplementary Table S1.

Fluorescence polarization assay for analysis of DNA binding

5′-FAM-labeled hemi-PT-DNA, labeled on one strand only, was synthesized and purified (Supplementary Table S1). Protein solutions were diluted serially using 2-fold dilutions (5 μM starting concentration, 16–20 dilutions) and mixed with a 5 nM final concentration of DNA probe in a Corning 3575 plate, using binding buffer of 20 mM Tris–HCl pH 8.0, 5% glycerol, 50 mM NaCl and 1 mM DTT. The mixture was incubated for 10 min at room temperature, and fluorescence polarization was measured at room temperature on a SpectraMax i3x (Molecular Devices) using 485/20 nm and 528/20 nm filters for emission and excitation, respectively. The dissociation constants (K_D) were calculated by fitting the experimental data (from two experimental replicates) to the following equation using GraphPad Prism software (version 6.0): [mP] = [maximum mP][C] /(K_D + [C]) + [baseline mP], and then the curve was replotted using percent saturation calculated as ([mP] – [baseline mP])/([maximum mP] – [baseline mP]), where mP is millipolarization and [C] is protein concentration. The binding experiments were performed under the same laboratory conditions.

Transformation efficiency assay

The pACYCDuet™-1 vector (PT⁻) and its derivative (PT⁺) carrying the dnd gene cluster from Salmonella enterica serovar Cerro 87 were introduced to E. coli BL21(DE3), and competent cells of the resulting strains were prepared using the standard calcium chloride protocol. Transformation frequency was determined by introducing 100 ng pET28a derivatives carrying scoMcrA or its mutant variants to the competent cells. The number of E. coli colonies in each experiment was determined by serial dilutions. Each experiment was repeated three times and the mean value of the transformation frequency was reported.

All-atom molecular dynamics simulation

The co-crystal structure of the SBD domain of ScoMcrA and the natural PT-DNA fragments (G_PSGCC) was used as a starting model to build up the nucleotide-mutant models of G_PSATC and G_PSAAC, and the protein-mutant models of E156R and E156R/D157R, with the modeling software package of Molecular Operating Environment v2018 (31). All the molecular dynamics simulations (MDs) were performed with the AMBER 16 software (30). For protein and DNA parts, Amber ff14SB and OL15 force field were used, respectively (32,33). The phosphorothioate force field employed these parameters developed by Mukherjee and Bhattacharyya et al. (34). The PROPKA algorithm determined the protonation of the SBD–DNA complex on the PDB2PQR web server (35). The protein-DNA complexes were then solvated within a cubic box and the TIP3P water model (36), in which the minimum distances between any protein atom and edges of the water box was set to be 12 Å. The systems were neutralized by adding appropriate numbers of Na⁺ and Cl⁻ ions. Long-range electrostatic interactions were calculated with the Particle-Mesh-Ewald (PME) method (37), and van der Waals interactions were truncated within 12 Å. The time interval was set as 2 fs, and the SHAKE (38) algorithm was used to constrain the bonds-connecting hydrogen atoms. The entire system was first minimized and heated up to 298 K before the production process. The CPPTRAJ tool implemented in the AMBER 16 software package was used for trajectory analyses, such as the popular root-mean square deviation (RMSD) and cluster analysis. Solvent accessible surface area (SASA) is a parameter that measures the fraction of the protein surface interacting with the solvent molecules. The term corresponding to the SASA was calculated through BIOVIA Discovery Studio (39).

Binding Free Energy Calculation

The binding free energy between PT-DNA and SBD was calculated by the MM/GBSA approach (40), using the following equations,

where ΔE_int is neglectable with the single-trajectory strategy. The nonpolar part of the solvation free energy (ΔG_SA) was calculated with the solvent-accessible surface area (SASA) through the LCPO algorithm (41), by using ΔG_SA = γ ⋅ SASA + β (the surface tension constants γ and β were set to 0.0072 and 0, respectively). The polar part of the solvation energy (ΔG_GB) was estimated using the Generalized Born (GB) model proposed by Onufriev et al. (GB^OBC1, igb = 2) (42). The ΔE_vdW, ΔEele, ΔG_GB and ΔG_SA terms were computed based on the 500 snapshots extracted from the last 20 ns MD trajectories. Each trajectory was calculated individually, and then all energies were analyzed statistically.

Non-covalent interaction (NCI) analysis

The independent gradient model (IGM) (43) and reduced density gradient (RDG) (44) analyses were carried out using Multiwfn 3.6 program (45). Molecular plots were visualized with the VMD 1.9.3 program (46).

The IGM analysis depends on the topological characteristics of the electron density, ρ. The IGM descriptor δg^inter is calculated as the difference between the first derivatives of electron density of the whole system and the fragments:

δg^inter > 0 indicates the presence of weak interactions and its magnitude denotes the interacting intensity.

The non-covalent interaction RDG method is an alternative method to reveal weak interlayer interactions (44), with a dimensionless form of electron density gradient norm function:

The sign of the second eigenvalue of the electron density Hessian matrix, sign(λ₂), was used in the RDG analyses to judge the attractive and repulsive interaction, that is, corresponding to negative and positive values of sign(λ₂) ρ, respectively.

RESULTS

Affinity of SBD_Spr for PT-DNA of varied sequence contexts

The sulfur modification-dependent REases SprMcrA and ScoMcrA use an SBD to recognize the DNA backbone phosphorothioate link of the R_P stereoisomer, which is adopted by the naturally occurring PT modifications in five DNA core sequence contexts (Table 1) in prokaryotes. ScoMcrA only recognizes G_PSGCC, whereas SprMcrA binds and shifts DNA of the sequences G_PSGCC, G_PSATC and G_PSAAC in EMSAs (21). To compare the affinity of SBD_Spr (aa 1–165 of SprMcrA) for PT-DNA of the five natural PT sequence contexts, a set of hemi-modified DNA duplexes, which differed from each other in the core sequence bearing the PT link in either the S_P or R_P configuration (Table 1) were assayed (Supplementary Figure S1). In agreement with the reported EMSA results (21), no S_P PT-DNA nor R_P PT-DNA in sequences of G_PSTTC or C_PSCA could be recognized by SBD_Spr, indicating that recognition of the PT link is coupled with interactions with surrounding nucleobase or phosphate groups. SBD_Spr showed the highest binding affinity for G_PSGCC, with a dissociation constant value (K_D) of 5.55 nM, followed by a K_D of 38.33 nM for G_PSATC and 94.67 nM for G_PSAAC (Table 1). By comparison, SBD_Sco showed a K_D of 102 nM for G_PSGCC (Table 2), 18.5-fold weaker binding than SBD_Spr had to the same DNA duplex.

Table 1.

Affinity of SBD_Spr for PT-DNA of different core sequences

PT-DNA		K D (nM)
GPSGCC	R P	5.6 ± 0.9
	S P	–*
GPSATC	R P	38 ± 8
	S P	–
GPSAAC	R P	95 ± 25
	S P	–
GPSTTC	R P	–
	S P	–
CPSCA	R P	–
	S P	–

*The binding affinity of SBD_Spr to substrate DNA was too weak, making the K_D value too large to be determined.

Table 2.

K _D (nM) value of SBD_Sco and SBD_Sco mutants for PT-DNA of different core sequences

	Hemi PT-DNA duplex
	GPSGCC	GPSATC	GPSAAC
SBDsco (wild type)	102 ± 12*	1091 ± 58	567 ± 37
E156D	112 ± 12	1322 ± 99	846 ± 74
E156Q	137 ± 18	1015 ± 49	618 ± 43
E156L	110 ± 12	791 ± 42	499 ± 37
E156K	130 ± 11	370 ± 14	297 ± 19
E156R	133 ± 21	346 ± 17	267 ± 15
E156R/D157R	123 ± 8	192 ± 12	183 ± 9

* K_D (dissociation constant, nM).

Structure of SBD_Spr complexes

To determine why SBD_Spr showed varying affinity for G_PSGCC, G_PSATC and G_PSAAC, as well as a much higher affinity for G_PSGCC, we determined the crystal structures of SBD_Spr in the presence of three hemi-PT-DNA oligonucleotides with the G_PSGCC, G_PSATC and G_PSAAC core sequences (PDB codes 7CC9, 7CCJ and 7CCD, respectively; Supplementary Table S2). The overall structures for the three complexes were similar with in the terms of sulfur coordination (Supplementary Figure S2) except for different base interaction (See below). To simplify the description of the SBD_Spr structure and facilitate comparative analysis with SBD_Sco-G_PSGCC, we here depict the details on one of the complexes, SBD_Spr–G_PSGCC as an example. The structure was determined by molecular replacement and refined to a resolution of 2.06 Å. The crystallographic asymmetric unit contained three protein molecules, with each of them associated with one molecule of hemi-PT-DNA (Supplementary Figure S3A). The three SBD molecules and their respective DNA molecules in a crystallographic unit were well aligned with each other (the value of root mean square error is less than 0.140 Å over 165 Cα atoms) (Supplementary Figure S3D). SBD_Spr comprised nine helices (α1–α9) and two β-sheets (βA and βB) (Figure 1A–C). SBD_Spr clearly did not make any contact with the DNA strand without the PT linkage. The sulfur atom from the DNA helical edge was positioned outward into the central bottom of a hydrophobic cavity that was formed by the side chains of five amino acid residues from the separated helices 2 and 4 (Figure 2A and B). The PT-DNA binds within a basic groove on the wedge-like surface of the SBD protein, leading to the mortise-and-tenon-like interactions (Figure 2A). The sulfur atom of the PT linkage was inserted into a cavity which formed by Y31, Q32, Y78, P79 and A82 through Van der Waals interactions; the phosphate groups flanking the PT linkage formed electrostatic bonds with R29, R73 and R85, as well as hydrogen bonds with Y31 and A101 (Figure 2C). As predicted, a single mutation within the five residues diminished the DNA binding affinity to varied extents, particularly with either of the two Y→A mutations, which almost abolished DNA binding affinity (Supplementary Figure S4, Table S3). In addition to interactions with the sulfur atom and phosphate backbone, the H102–G103–D104 motif of loop A5 inserted into the PT-DNA major groove to form five hydrogen bonds (H-bond) with bases of G_PSGCC core sequence (Figures 1B, 2C and 3).

Figure 1.

SBD of the PT-dependent restriction endonucleases SprMcrA and ScoMcrA. (A, B) Two views of the SBD of SprMcrA (SBD_Spr) binding PT-DNA specifically. Loop 12, loop 34 and loop A5, which interact with DNA, are indicated by red arrows. The side chains of His102 and Asp104 (colored in purple) are located in the DNA major groove. The green ball in DNA molecule denoted the sulfur atom. (C) Sequence alignment of the SBD of SprMcrA (SBD_Spr) and ScoMcrA (SBD_Sco). The conserved residues forming the sulfur atom-binding pocket are marked with yellow triangles. Secondary structure elements of SBD_Spr and SBD_Sco are numbered according to crystal structures (PDB code: 7CC9 and 5ZMO). The loops that interact with DNA are marked by green boxes. Alignment was generated with ESPript.

Figure 2.

Details of the SBD_Spr–DNA interactions. (A) The SBD_Spr binds specifically to G_PSGCC. Residues that interact with DNA are colored as follows: Tyr31, Gln32, Tyr78, Pro79 and Ala82, which form the sulfur atom binding pocket, are in yellow; His102, Gly103 and Asp104, which recognize DNA bases, are in purple; and Arg29, Arg73 and Arg85, which interact with phosphates through electrostatic interactions, are in blue. (B) Sulfur atom-binding pocket on SBD_Spr formed by Tyr31, Gln32, Tyr78, Pro79 and Ala82. (C) Schematic summary of the interactions between SBD_Spr and PT-DNA, mC represented the main chain of amino acid.

Figure 3.

Comparison of HGD motif of SBD_Spr interaction with three DNA core sequences of (A) G_PSGCC, (B) G_PSATC and (C) G_PSAAC. SG5 and SA5 represents the nucleoside G5 and A5 with PT modification. The base contact motif HGD of SBD_Spr in the three complexes was shown in cyan, purple and green, respectively.

Comparison of the structure of SBD_Spr and SBD_Sco complexed with G_PSGCC

Generally, the overall structure of the SBD_Spr monomer, including the sulfur-binding cavity, was similar to SBD_Sco except for three flexible loops that swayed differently (Supplementary Figure S5). Among them, loop 12 made no contact with DNA substrates in either structure. Loop 34 was three amino acids longer in SBD_Spr than in SBD_Sco and made contacts with the DNA phosphate backbone. The location of loop A5 in SBD_Spr was shifted by four amino acids from the equivalent loop 56 in SBD_Sco (Figure 1C); however, both of these loops were inserted into the major groove of the DNA substrates and contacted the DNA bases (Figure 1A, B). The sequence and spatial arrangement of these residues involved in sulfur coordination were well aligned with the equivalent residues of SBD_Sco, but differed slightly by the presence of an additional electrostatic bond with the guanidine group of R117 in SBD_Sco (Supplementary Figure S6). The S_P oxygen symmetric to sulfur was stabilized by two hydrogen bonds with Q32 and A22 in SBD_Spr whereas it only bonded to the imino group of R117 in SBD_Sco. The Q32A mutation in SBD_Spr caused an ∼50-fold decrease in binding affinity, but the equivalent mutation of R117A or R117G in SBD_Sco completely abolished the affinity for PT-DNA, possibly due to the loss of three bonds between R117 and the sulfur and oxygen atoms.

Except for the electrostatic interaction between the phosphate group of G⁴ and R85, the remaining four bonds lack equivalents in the structure of the SBD_Sco complex (20). Therefore, the striking structural difference between the two complexes with respect to the interaction with the phosphate backbone lies in the lack of any interaction with the phosphate group of C⁶, immediately downstream of the phosphorothioate in SBD_Sco. On the contrary, in SBD_spr, Y31 also makes a hydrogen bond to the fifth DNA phosphate in addition to coordination with the sulfur atom (Figure 2C).

Base contact by SBD_Spr determines the variation in binding affinity

As mentioned above, SBD_Spr displayed varied affinity to PT–DNA of different core sequences (Table 1). In three SBD_Spr co-crystal structures, the H102–G103–D104 motif all inserted into the DNA major groove to make contacts with bases, but the numbers of H-bonds were slightly different (Figure 3). In all structures, the ND1 atom of H102 formed H-bond with O6 atom of G⁴, and the OD2 atom of D104 bonded to the N4 atom of C⁷. It's worth noting that the ND1 atom of H102 also formed H-bond with N7 atom of G⁴ in G_PSGCC and G_PSAAC sequences, while this H-bond was not existing in SBD_Spr-G_PSATC complex. When the central SG⁵C⁶ are changed to SA⁵A⁶ or SA⁵T⁶, the H-bonds patterns formed by the central bases showed some differences in three complexes. The N atom in the main chain of G103 bonded to N7 atom of SG⁵ from G_PSGCC sequence and N7 atom of SA⁵ from G_PSATC and G_PSAAC sequences. The carbonyl O atom of G103 formed an additional H-bond with N4 atom of base C⁶ in SBD_Spr-G_PSGCC complex. SA⁵ showed a significant deflection in G_PSATC sequence, compared with G_PSGCC and G_PSAAC sequences, leading to formation of another H-bond between the ND1 atom of H102 with the N6 atom of SA⁵. In conclusion, HGD motif formed five H-bonds in SBD_Spr–G_PSGCC complex while four H-bonds in SBD_Spr–G_PSATC and SBD_Spr–G_PSAAC complexes, which explained why SBD_Spr showed a highest affinity for G_PSGCC.

The base recognition pattern by HGD motif in complexes of SBD_Spr–G_PSAAC and SBD_Spr–G_PSATC is different (Figure 3). What's more, close comparison of two structures revealed that the binding of the G_PSAAC released the methyl group of T6′ on the complementary strand from the binding site, and converts the weak non-bonded interaction to unfavored thymine methyl-solvent accessibility (Supplementary Figure S7). In the case of G_PSATC, the percent solvent accessibility of T6 methyl group was calculated to be only 10–15, corresponding to fully buried with the SBD-DNA interface. The multiple C–H…O contacts between the methyl and Y78 were believed to be attractive for G_PSATC (47). In the case of G_PSAAC, the T6′ methyl group was fully exposed to solvent and lack of specific interaction with SBD protein. Difference in the weak non-bonded interaction between G_PSATC and G_PSAAC results in a lower K_D values for G_PSATC than G_PSAAC bound by SBD_Spr.

To evaluate the contribution of these interactions with the bases to the DNA binding affinity, the three aa residues HGD were independently mutated, and the binding affinity of the resulting mutated proteins to hemi-PT-DNA of 5′-GGCG_PSGCCC-3′ was measured by fluorescence polarity (Supplementary Table S3). The H102A and D104A mutants showed a 370-fold and 25-fold decrease, respectively, in binding affinity compared with wild-type protein, demonstrating that base contact constitutes an important component of the total affinity for PT-DNA by ensuring the formation of a stable DNA/protein complex. Unexpectedly, the G103A mutation almost abolished the affinity for PT-DNA as evidenced by the strikingly increased K_D value of >9000 nM (Supplementary Table S3). The G103A mutation introduced an additional C-C side chain, which increased the main chain rigidity and affecting the hydrogen bonding network of base G⁵ and C⁶, leading to drastic decrease in binding affinity.

Opposite interactions with PT-DNA by loop 34 of SBD_Spr and SBD_Sco

When the structures of SBD_Spr–G_PSGCC and SBD_Sco–G_PSGCC were compared, a striking DNA strand distortion at the two phosphodiester bonds proximal to the 3′ terminus of the PT-DNA strand was observed in the SBD_Sco–G_PSGCC complex. Phosphorus atoms of the seventh and eighth bases in the PT-modified strand were extruded by 3.5 and 5.0 Å relative to those in the SBD_Spr–G_PSGCC structure. (Figure 4A). Compared with SBD_Sco, SBD_Spr possesses a longer loop 34, containing the three positively charged residues R69, R73, and R75, which constitute a local positive interface with DNA wherein R73 bonds to the phosphate group of C⁶ (Figure 4B, D). By contrast, the corresponding interface of the SBD_Sco loop34 features two tandem acidic residues, E156 and D157, and a spatially adjacent D160 (Figure 4C, E). These residues form a negatively charged surface area, which is repulsive towards the DNA phosphate backbone and may account for the distortion of the DNA double helix structure in the SBD_Sco–G_PSGCC complex.

Figure 4.

Comparison of SBD_Spr and SBD_Sco interactions with PT-DNA. (A) Superimposition of SBD_Spr loop34 (cyan) with DNA (orange) and SBD_Sco loop34 (grey) with DNA (yellow). Phosphorus atoms of the seventh and eighth bases in the PT-modified strand of SBD_Sco-G_PSGCC were extruded by 3.5 and 5.0 Å relative to those in the SBD_Spr–G_PSGCC. (B) Arg69, Arg73 and Arg75 of SBD_Spr loop 34 form a positive interface with the DNA. (C) Glu156, Asp157 and Asp160 of SBD_Sco loop 34 and helix 3 form a negatively charged surface area to DNA. (D, E) The surface charge of (D) SBD_Spr and (E) SBD_Sco. The surface charge distribution at neutral pH is displayed with blue for positive, red for negative, and white for neutral. (F) Influence of mutations in E156 on the ability of SBD_Sco to bind G_PSGCC, G_PSATC and G_PSAAC in EMSAs. N, no protein added to the EMSA.

Ability of SBD_Sco–E156R/D157R to bind PT-DNA of G_PSAAC and G_PSATC

Given that SBD_Sco can only bind to G_PSGCC, and that SBD_Spr has maximum affinity for G_PSGCC, we hypothesized that the repulsive force exerted by the negative interface of SBD_Sco weakened its overall affinity for PT-DNA, leading to the failure to recognize G_PSAAC or G_PSATC, although this repulsive force was not sufficient to disrupt the most stable complex formed with G_PSGCC.

To test this hypothesis, E156 of SBD_Sco, structurally equivalent to R73 of SBD_Spr, was mutated into basic (R and K), neutral (L and Q), and acidic (D) residues. Affinity quantification of each E156 mutant by fluorescence polarization assay showed that the mutants containing the R, K or L substitutions all showed an increased affinity for G_PSAAC and G_PSATC relative to the wild-type protein. In particular, the E156R mutant displayed the most significant increases in DNA binding affinity for G_PSATC and G_PSAAC by, respectively, ∼3-fold and ∼2-fold (Table 2, Figure 4F, Supplementary Figure S8). The double-mutation protein, E156R/D157R, showed further increases in DNA binding affinity for G_PSATC and G_PSAAC in EMSA (Figure 5A), which were quantified to be ∼5.7-fold and ∼3-fold increases for G_PSATC and G_PSAAC, respectively (Table 2). Unfortunately, we were unable to purify the triple-mutation protein, E156R/D157R/D160R, probably because the significant decrease in protein expression. However, mutations of the three acidic residues were constructed in the full-length ScoMcrA, and the in vivo nuclease activities of the mutants were analyzed by comparing the transformation efficiency of their coding DNA into a PT and non-PT E. coli host (Figure 5B). In agreement with the EMSA results for the SBD mutants, the uptake efficiency of scoMcrA_E156R by the PT host was 2.5-fold less than by the non-PT host, while that of other two single-mutant genes had no significant difference in the PT and non-PT hosts. In parallel, the transformation efficiency of scoMcrA_E156R/D157R was 500-fold less with PT E. coli than with non-PT E. coli (Figure 5B), implying that the double-mutation protein acquired restriction activity for G_PSAAC DNA, but kept the ability to discriminate the unmodified DNA. However, the triple-mutant gene did not show distinctive transformation efficiency between the PT and non-PT E. coli hosts, but rather exhibited decreased efficiency with both hosts.

Figure 5.

Sequence specificity of ScoMcrA mutants. (A) Ability of SBD_Sco mutants to bind G_PSGCC, G_PSATC and G_PSAAC in EMSAs. N, no protein added to the EMSA; WT, the wild-type SBD_Sco; E156R, SBD_Sco–E156R mutant; E156R/D157R, SBD_Sco–E156R/D157R mutant. (B) Uptake efficiency of scoMcrA and its mutants by the PT⁻ host and PT⁺ host. The PT⁺ host contains the expression vector with the dnd gene cluster from Salmonella enterica, which encodes the ‘writer’ proteins for phosphorothioation of G_PSAAC /G_PSTTC. Transformation efficiency obtained with the dnd host (PT⁺) and the negative control host (PT⁻) is indicated by black bars and white bars, respectively.

To further explore the key structural parameters that affect the binding of PT-DNA and protein, we performed multiple 100 ns MD simulations of the wild-type complex, as well as of the single mutation (E156R) and double mutation (E156R/D157R) complexes for SBD_Sco–G_PSATC and SBD_Sco–G_PSAAC, which were built and mutagenized through Molecular Operating Environment. The most populated conformations sampled during our simulations that contain the interaction regions were chosen for RDG analysis. The calculated RDG isosurfaces with BGR color scales representing sign(λ₂) ρ values are given in Supplementary Figure S9 for E156R and E156R/D157R in SBD_Sco-G_PSATC and SBD_Sco-G_PSAAC. In E156R, two hydrogen bonds, N7_G⁷⋅⋅⋅NH1_R156 and O6_G⁷⋅⋅⋅NH2_R156, were strong. When we introduced a double mutation (E156R/D157R), two newly formed hydrogen bonds (HH12_R157⋅⋅⋅O6_G⁷ and HH22_R156⋅⋅⋅O2P_C⁶) were observed, similar to the case of E156R. Additionally, van der Waals interactions were also observed between the adjacent HH22_R157 and O6_G⁷ and N7_G⁷, indicated by the green color of the RDG isosurfaces. The gain of G_PSATC and G_PSAAC interactions with both R156 and R157 might be crucial to triggering the changes leading to the acquisition of the enhancement of non-covalent interaction. Replacement of E156 and D157 with arginine introduced hydrogen bond interactions as well as van der Waals interactions, which significantly strengthened the binding, resulting in a concerted interplay of interactions between the SBD and PT-DNA.

DISCUSSION

Recognition of PT-DNA by the SBD of the type IV REase ScoMcrA is not only phosphorothioate-dependent but also DNA sequence-specific as this enzyme only recognizes PT-DNA of the G_PSGCC core in vivo and in vitro, whereas it does not bind to the PT-DNA of other four core sequences found in prokaryotes. However, five SBD_Sco homologs, including SprMcrA, generally display a more relaxed sequence specificity in target DNA selection. Although both SBD_Spr and SBD_Sco clearly shifted the G_PSGCC DNA duplex in EMSAs, affinity quantification showed that the former had an 18.2-fold higher affinity than the latter did, with SBD_Spr also showing a 28.7-fold and 6-fold higher affinity for G_PSATC and G_PSAAC, respectively (Tables 1 and 2). These differences in binding affinity between the two SBD domains lead to a distinctive presence or absence of in vivo restriction activity. For example, ScoMcrA can restrict the uptake of dnd gene clusters generating the G_PSGCC modification (20) but not those generating the GAAC/GTTC modifications (Figure 5B). In contrast, SprMcrA can block the establishment of PT modifications at G_PSAAC/G_PSTTC and G_PSGCC sites as it showed an overall higher affinity for PT-DNA compared to ScoMcrA. Through comparative analysis, we attributed this difference in binding affinity to the reverse charge of loop 34 in both structures, which functions like a switch by changing between positive and negative electric charges in the different structures. Mutations of negatively charged amino acids into positively charged ones on loop 34 of SBD_Sco significantly enhanced the binding affinity for PT-DNA. For example, the E156R/D157R mutation conferred SBD_Sco with the ability to bind to G_PSAAC and G_PSATC, and thus conferred ScoMcrA with in vivo restriction activity for dnd encoding G_PSAAC/G_PSTTC (Figure 5B). This structural switch offers us an opportunity to engineer a flexible or stringent sequence specificity for a given SBD.

In our study, the SBD_Sco–E156R and SBD_Sco–E156R/D157R mutants had significant increases in binding affinity for G_PSATC and G_PSAAC when compared with the wild-type SBD_Sco (Table 2). The MD simulations showed that R156 and R157 participate in van der Waals interactions and hydrogen-bond interactions with C⁶ and G⁷ of PT-DNA with G_PSATC and G_PSAAC core sequences, resulting in the higher binding affinity compared to the wild-type SBD_Sco. However, the binding affinity of these two mutants for G_PSGCC showed a slight decrease in comparison to the wild-type protein, in contrast to the increased affinities for G_PSATC and G_PSAAC. The superposition of the mutants and wild type structures after MD simulations gave a root mean square deviation (RMSD) value of 0.636 Å by using backbone atoms (Cα), indicating the mutations do not lead to vastly structural changes in the MD simulations (Supplementary Figure S10). Next, the binding affinity of G_PSGCC and SBD_Sco were carefully examined to understand the geometrical disturbance of E156R/D157R mutation with the MM/GBSA method (40). As shown in Supplementary Table S4, the ΔΔG_binding value for PT-DNA binding SBD_Sco–E156R/D157R was positive (0.9 kcal/mol), suggesting that the mutation slightly weakened the binding affinity, consistent with the experimental observation (Table 2). It is noticed that deformability of the DNA structure may contribute to the sequence specificity (48). As the conclusions of MD simulations, R156 and R157 are not directly involved in influencing binding interaction with C⁶ and G⁷ in G_PSGCC sequence, however, they may affect the orientation of other residues that are involved in direct interaction with PT-DNA. We speculate that will lead to the twisting of DNA, which then results in an imperfect match of the hydrophobic pocket with the R_P sulfur atom, thus reducing the affinity. These interactions ultimately lead to an overall decrease in the binding affinity of mutant SBD_Sco–E156R/D157R to G_PSGCC. Interestingly, the SBD_Sco–E156R/D157R mutant gained the ability to bind with the S_P stereoisomers of G_PSGCC (Supplementary Figure S11) probably because the twisting of the G_PSGCC strand by the E156R/D157R mutation positioned the sulfur of S_P within the sulfur-coordination cavity.

SBD homologs are widely represented in at least 1059 sequenced species from 14 phyla of bacteria (20). In addition to SBD_Sco, four SBD homologs, including SBD_Spr, displayed flexibility in the selection of substrate PT-DNA with different core sequences (20). It is notable that loop 34 is rich in basic amino acids in four of the SBDs (Supplementary Figure S12). With its acidic amino acids, loop 34 of SBD_Sco is unique among SBD homologs, which may be related to the unique domain composition of SBD-SRA-HNH for ScoMcrA. Multiple DNA recognition domains of ScoMcrA result in reduced ability to distinguish between modified and non-modified DNA substrates, in turn resulting in nonspecific cleavage activity. In order to maintain the specificity of cleavage activity and low toxicity, the distribution of positive charges on the surface may have become reduced in ScoMcrA during evolution. Consequently, ScoMcrA can only recognize and restrict G_PSGCC, the most common core sequence of PT-DNA in Streptomyces, with flexibility lost in the selection of substrate PT-DNA with different core sequences. Overall, our study illustrates structural features that impact the recognition of PT-DNA by SBDs of type IV restriction enzymes.

DATA AVAILABILITY

Atomic coordinates and structure factor for the sulfur-binding domain (SBD) from full-length Streptomyces pristinaespiralis endonuclease SprMcrA in complex with 5′-GGCG_PSGCCC-3′, 5′-GATG_PSATCC-3′ and 5′-GGCG_PSAACGTG-3′ have been deposited with the Protein Data Bank under accession numbers 7CC9, 7CCJ and 7CCD, respectively.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

National Natural Science Foundation of China [31770070, 31970041, 31670106, 31872627]; Chinese Postdoctoral Science Foundation [2019M661493]; National Key Research and Development Program of China [2018YFA0901900]. Funding for open access charge: National Natural Science Foundation of China [31770070]; Chinese Postdoctoral Science Foundation [2019M661493].

Conflict of interest statement. None declared.