
==== Front
Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

52130
10.1038/s41467-024-52130-x
Article
Burkholderia cenocepacia epigenetic regulator M.BceJIV simultaneously engages two DNA recognition sequences for methylation
http://orcid.org/0000-0002-5835-4683
Quintana-Feliciano Richard 1
Kottur Jithesh jitheshkottur@iav.res.in

12
http://orcid.org/0000-0002-8930-6887
Ni Mi 3
Ghosh Rikhia 1
Salas-Estrada Leslie 1
Ahlsen Goran 4
http://orcid.org/0000-0003-4270-7242
Rechkoblit Olga 1
Shapiro Lawrence 4
http://orcid.org/0000-0002-4382-8276
Filizola Marta 1
http://orcid.org/0000-0002-2462-9124
Fang Gang 3
http://orcid.org/0000-0003-4389-0173
Aggarwal Aneel K. aneel.aggarwal@mssm.edu

1
1 https://ror.org/04a9tmd77 grid.59734.3c 0000 0001 0670 2351 Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, New York USA
2 Department of Antiviral Drug Research, Institute of Advanced Virology, Thiruvananthapuram, Kerala India
3 https://ror.org/04a9tmd77 grid.59734.3c 0000 0001 0670 2351 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, New York USA
4 https://ror.org/00hj8s172 grid.21729.3f 0000 0004 1936 8729 Department of Biochemistry and Molecular Biophysics Columbia University Vagelos College of Physicians and Surgeons, New York, NY USA
7 9 2024
7 9 2024
2024
15 783929 10 2023
27 8 2024
© The Author(s) 2024
2024
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Burkholderia cenocepacia is an opportunistic and infective bacterium containing an orphan DNA methyltransferase called M.BceJIV with roles in regulating gene expression and motility of the bacterium. M.BceJIV recognizes a GTWWAC motif (where W can be an adenine or a thymine) and methylates N6 of the adenine at the fifth base position. Here, we present crystal structures of M.BceJIV/DNA/sinefungin ternary complex and allied biochemical, computational, and thermodynamic analyses. Remarkably, the structures show not one, but two DNA substrates bound to the M.BceJIV dimer, with each monomer contributing to the recognition of two recognition sequences. We also show that methylation at the two recognition sequences occurs independently, and that the GTWWAC motifs are enriched in intergenic regions in the genomes of B. cenocepacia strains. We further computationally assess the interactions underlying the affinities of different ligands (SAM, SAH, and sinefungin) for M.BceJIV, as a step towards developing selective inhibitors for limiting B. cenocepacia infection.

Crystal structures of DNA methyltransferase M.BceJIV in complex with DNA and sinefungin reveal an unusual mode of DNA binding and methylation, wherein each M.BceJIV monomer contributes to the recognition of two DNA sequences.

Subject terms

Bacterial structural biology
X-ray crystallography
DNA methylation
https://doi.org/10.13039/100000002 U.S. Department of Health & Human Services | National Institutes of Health (NIH) R35-GM131780 R35GM13965 Fang Gang Aggarwal Aneel K. U.S. Department of Health & Human Services | National Institutes of Health (NIH)issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Burkholderia cenocepacia is an opportunistic and infective bacterium, a member of the Burkholderia cepacia complex (Bcc), that often leads to severe illness in immunocompromised patients1. Antibiotics in clinical use against Bcc infection include the aminoglycoside Tobramycin and the β-lactams Aztreonam and Doripenem. B. cenocepacia’s ability to form biofilms and its antibiotic resistance highlights the importance of new approaches to counter its infectivity. Although bacterial DNA methyltransferases (MTases) are largely associated with restriction-modification (R-M) systems for defense against phages2, they can also play roles in gene regulation3–8 and serve as potential drug targets5,6,9.

A DNA MTase was recently identified in B. cenocepacia, BCAM0992 (here referred to as M.BceJIV), deletion of which compromises the motility of the bacterium10. M.BceJIV is a 32 kDa MTase, part of the trp operon, and potentially under the same regulation. It recognizes the GTWWAC motif (where W can be an adenine or thymine) and methylates the N6 of the adenine at the fifth base position (GTWWAC)10. M.BceJIV is an orphan MTase, without a cognate restriction endonuclease, and is strongly conserved across the diverse species and strains of the Burkholderia genus, indicating its possible importance in the physiology of Burkholderia species10. Deletion of M.BceJIV leads to the upregulation of several genes with GTWWAC sites in their promoters, including the DNA helicase gene (rep) that has been shown to play a role in swimming motility in Escherichia coli and may be correlated to the dysfunction in motility observed in M.BceJIV mutants10.

As an orphan MTase, M.BceJIV joins a growing list of bacterial MTases with roles outside of R-M and defense against foreign DNA7. Classical examples of orphan MTases are Dam in Escherichia coli, CcrM in Caulobacter crescentus, and Dcm in Escherichia and Salmonella genera, functioning in DNA replication/repair, cell cycle regulation and the expression of specific genes, respectively7. The recent discovery of an orphan MTase, CamA, in Clostridioides difficile with roles in the sporulation and motility of the bacterium highlights the therapeutic opportunities in targeting regulatory MTases in pathogenic bacteria6,8. We present here crystal structures of M.BceJIV/DNA/sinefungin complex that, unexpectedly, shows not one, but two DNA substrates bound to the M.BceJIV dimer. Remarkably, each monomer contributes to the recognition of two recognition sequences. As a key factor in B. cenocepacia’s motility, the M.BceJIV structure offers new opportunities in the design of therapeutics to control bacterial infection.

Results

Structure determination

M.BceJIV was cocrystallized with a pair of complementary 14-base-pair oligonucleotides with overhanging 5’-ends (A or T). The resulting DNA duplex contains a single M.BceJIV recognition site (GTTAAC). The cocrystals were obtained in the presence of sinefungin (in place of the natural cofactor SAM) and diffracted to ~ 2.1 Å resolution with synchrotron radiation. The cocrystals belong to space group I41 with unit cell dimensions of a = b = 137.7 Å, c = 167.3 Å, and α = β = γ = 90o and contain two M.BceJIV dimers, four DNA duplex substrates, and four sinefungin molecules in the crystallographic asymmetric unit (Supplementary Table 1). The refined model consists of four M.BceJIV subunits (A, residues 30–279; B, residues 30–278; C, residues 30–279; D, residues 31–277), four DNA duplex substrates (DNA1, DNA2, DNA3, and DNA4), four sinefungin molecules, and 565 solvent molecules. M.BceJIV subunits A and B form a complex with DNA1 and DNA2 (complex 1), whereas subunits C and D form a complex with DNA3 and DNA4 (complex 2). The two complexes are very similar. The electron density for complex 1 is better defined than that for complex 2, and we describe the structure of complex 1 hereafter.

In addition, we determined the structures of M.BceJIV with other variations of the GTWWAC recognition sequence, namely GTATAC, GTAAAC, and GTTTAC. As the structure with GTTAAC (above), the cocrystals with these sequence variants belong to space group I41 with unit cell dimensions of a = b = 137.7 Å, c = 166.6–167.8 Å, and α = β = γ = 90o and contain two M.BceJIV dimers, four DNA duplex substrates, and four sinefungin molecules in the crystallographic asymmetric unit (Supplementary Figs. 1, 2 and supplementary Table 1). M.BceJIV binds to these sequence variants very similarly to that observed with GTTAAC. We describe here primarily the structure of M.BceJIV with the GTTAAC recognition sequence and refer to the other variants as needed.

Overall architecture

The M.BceJIV monomers A and B form a two-fold symmetric dimer that interacts extensively with two DNA duplexes (DNA1 and DNA2) containing the GTTAAC recognition motifs (Fig. 1). The target adenine (GTTAAC) flips out of each DNA duplex and enters the catalytic pockets of monomers A and B containing a sinefungin molecule (Fig. 1). Each M.BceJIV monomer contains the nine motifs (I–VIII and X) characteristic of amino MTases11 that together form an αβα “hub” consisting of a central eight-stranded β-sheet (β1-β8) with helices (αA, αB, αC, αF, αG and αH) on either side, from which the target recognition domain (TRD, composed of two short α-helices, αD and αE), protrudes outwards (Fig. 1). The TRD is a region of DNA MTases responsible for DNA recognition. Based on the linear order of the motifs, M.BceJIV belongs to the β class of amino MTases11, wherein the TRD (residues 167 to 194) is inserted between the N-terminal (IV–VIII) and the C-terminal (X and I–III) motifs. Motif I contains the FxGxG sequence for binding the cofactor SAM (or sinefungin in our case) and motif IV contains the (D/N)PP(Y/F) sequence for catalysis. The dimer interface between monomers A and B is extensive (~ 1738 Å2), mediated mostly by helix αC, strands β4–β6, and loop-45 (between strands β4 and β5) from each monomer.Fig. 1 Overall structure of M.BceJIVΔ29-DNA-sinefungin complex.

M.BceJIV forms a homodimer complex with two DNAs and two sinefungin molecules. Monomer A (magenta) receives the adenine to be methylated from DNA1, but its TRD reaches over to DNA2 and, conversely, monomer B (cyan) receives the adenine to be methylated from DNA2, but its TRD extends over to DNA1. Helices are labeled from αA to αH, and strands are labeled from β1–β8. Sinefungin is shown in a stick representation. The DNA substrate used for crystallization is shown, and the recognition sequence is boxed and numbered.

Monomer A receives the adenine to be methylated from DNA1, but its TRD reaches over to DNA2 and, conversely, monomer B receives the adenine to be methylated from DNA2, but its TRD extends over to DNA1 (Fig. 1 and Fig. 2a, b). Thus, each monomer contributes to the recognition of its own target DNA via its loop-45 and loop-6F (between strand β6 and helix αF) and to the second DNA via its TRD (Fig. 2a, b). The TRD recognizes the DNA through the major groove, whereas loop-45 and loop-6F recognize the DNA through the minor groove. This rather unique mode of DNA recognition, whereby each M.BceJIV monomer contributes to the recognition of two DNA sequences, is, to our knowledge, unprecedented for a DNA MTase.Fig. 2 Structure of M.BceJIVΔ29 monomers bound to DNA and sinefungin, and the electron density for DNA and sinefungin molecules.

Structure of monomer A bound to DNA1 and sinefunginA (a) and of monomer B bound to DNA2 and sinefunginB (b) showing DNA distortions and the TRDs. Each monomer contributes to the recognition of its own methylation-target DNA via its loop-45 and loop-6F through the minor groove and to the recognition of a separate DNA via its TRD through the major groove. 2Fo-Fc map contoured at 1.5 σ is shown for DNA1 and sinefunginA (c) and for DNA2 and sinefunginB (d). The recognition sequence is labeled.

The DNAs (DNA1 and DNA2) are severely distorted from B-DNA conformation over the recognition sequence (GTTAAC) (Fig. 2c, d). In addition to the target adenine (GTTAAC, A5) being flipped out of the DNA helix, the cytosine 3’ to it (GTTAAC, C6) is also rotated out of the helix by ~ 90o, and the base pairs at the third and fourth positions (GTTAAC) are highly buckled at angles of ~ 28o and 51o, respectively (Fig. 2). The major groove over the recognition sequence widens to ~ 24 Å (from ~ 19 Å in B-DNA) to accommodate the TRD and the minor groove widens to ~ 18 Å (from ~ 12 Å in B-DNA) to accommodate loop-45 and loop-6F. Strikingly, residues 136 to 138 from loop-45 are lodged between the unpaired bases of the fifth and sixth base pairs (GTTAAC) and drive the adenine and the cytosine to their extrahelical positions (Fig. 3a).Fig. 3 Molecular basis of target (DNA1) recognition.

a The first base pair (G1:C1) is specified by contacts with Gln207 of loop-6F of monomer A and Arg180 of TRDB; the second base pair (T2:A2) makes contact with His205 of loop-6F of monomer A and van der Waals contacts with Arg180 of TRDB; the third base pair (T3:A3) is specified by contacts with His205 and Arg203 of loop-6F of monomer A and van der Waals contacts with Leu182 and Phe183 of TRDB; the fourth base pair (A4:T4) makes contacts with Arg203 and van der Waals contacts with Met136 of monomer A (from loop-45) and van der Waals contacts with Trp188 of TRDB; at the fifth base pair (A5:T5), the thymine opposite the adenine to be methylated is specified by contacts with Gly137 of loop-45 that embeds in place of the everted fifth adenine and sixth cytosines of the target strand, and van der Waals contacts with Trp188 of TRDB; at the sixth base pair (C6:G6), the guanine opposite to the everted cytosine makes contacts with Gly137 of loop-45 of monomer A. b A close-up view of the flipped-out adenine that is accommodated in the catalytic cleft of monomer A and makes contacts with Asp59, Pro60 and Tyr62 of the conserved DPPY motif and with Tyr68 and Lys218 of monomer A. c A close-up view of the flipped-out cytosine accommodated outside the catalytic cleft and makes contacts with Asp148, His147, Thr106, Trp107, and Gln108 of monomer A. Monomer A is colored as magenta and the TRDB is colored as cyan.

The structures of M.BceJIV with variations of the GTWWAC recognition sequence, namely GTATAC, GTAAAC, and GTTTAC, reveal very similar binding modes to that observed with GTTAAC, with minimal changes in the overall structures. The RMSD between the GTTAAC structure and the variants GTATAC, GTAAAC, and GTTTAC are 0.144 Å (for 478 Cα atoms), 0.134 Å (for 478 Cα atoms), and 0.183 Å (for 482 Cα atoms), respectively. These results indicate that the unusual mode of M.BceJIV binding to two DNA sequences is not dependent on variations in recognition sequence but a unique feature of M.BceJIV (Supplementary Fig. 3).

DNA recognition

For DNA1, the TRD of monomer B inserts into its major groove, and loop-45 and loop-6F of monomer A insert into the minor groove. For DNA2 it is the converse, wherein TRD of monomer A inserts into its major groove and loop-45 and loop-6F of monomer B insert into the minor groove (Fig. 2a, b). Thus, each monomer recognizes both DNAs. Considering DNA1 (and the same applies to DNA2), the TRD contributes to the recognition of the first five of the six base-pairs of the recognition sequence (GTTAAC) (Fig. 3a). Specifically, Arg180 of the TRD makes bidentate hydrogen bonds with the O6 and N7 atoms of the outer guanine (GTTAAC) and van der Waals contacts with the C5 methyl group of the second thymine (GTTAAC), and Leu182 and Phe183 make van der Waals contacts with the C5 methyl of the third thymine (GTTAAC) (Fig. 3a). The fourth (A:T) and fifth (A:T) base pairs of the recognition sequence are specified by Trp188 that impinges on the C5 methyl groups of the thymines of these base-pairs (Fig. 3a). Loop-6F makes contacts through the minor groove, wherein Gln207 makes a hydrogen bond with the N2 amino group of the outer guanine (GTTAAC), His205 makes hydrogen bonds with the O2 atoms of the second and third thymines (GTTAAC), and Arg203 makes hydrogen bonds to the O2 atom of the third thymine (GTTAAC) and to the N3 atom of the fourth adenine (GTTAAC) (Fig. 3a). A short segment of loop-45 (Met136-Gly137-Gly138) embeds between the everted adenine and cytosine bases (GTTAAC) on the template strand and the partner thymine and guanine bases on the non-template strand (Fig. 3). Specifically, the main chain amide and carbonyl groups of Gly137 make direct hydrogen bonds with the thymine and guanine bases, respectively, facilitating their identity and acting as a wedge to evict the partner adenine and cytosine bases (GTTAAC) out of the DNA helix (Fig. 3). The flipped-out adenine of DNA1 enters the catalytic cleft of monomer A (where sinefungin is bound) and makes a series of specific contacts with residues Asp59, Pro60 and Tyr62 of the DPPY motif, conserved among amino MTases (described below). This adenine also makes additional polar interactions with Tyr68 and Lys218 (Fig. 3b). The flipped-out cytosine lies outside of the catalytic cleft and its entire Watson-Crick face is engaged in hydrogen bonds with Thr106, Trp107, Gln108, His147, and Asp148 (Fig. 3c). In addition, Met136 of Loop-45 impinges on the A:T base pair at the fourth position (GTTAAC) and contributes to the highly buckled state (~ 51o) of this base pair (Fig. 3a, A4:T4). Overall, M.BceJIV recognizes DNA through a combination of base-specific contacts and distortion of the DNA. That is, the free energy to adopt the distorted configuration of the DNA with evicted and buckled bases (Fig. 2c, d) is favored by the excess of A:T base pairs within the recognition sequence (GTWWAC).

We also resolved structures of M.BceJIV with the sequence variants GTATAC, GTAAAC, and GTTTAC. M.BceJIV binds to these sequences in a very similar manner to that observed with GTTAAC with only minor differences arising from the manner in which Arg203 interacts with the one or two base-pair changes. From above, for GTTAAC, Arg203 makes bidentate hydrogen bonds to the O2 atom of the third thymine (GTTAAC) and to the N3 atom of the fourth adenine (GTTAAC) (Fig. 3a). GTATAC differs from GTTAAC at positions 3 and 4, resulting in Arg203 making bidentate hydrogen bonds with the O2 atom of the thymine of the A:T base pair at position 3 (GTATAC) and the O2 atom of the thymine at the position 4 (GTATAC) (Supplementary Fig. 3a). GTAAAC differs from GTTAAC at position 3, resulting in Arg203 making bidentate hydrogen bonds to the O2 atom of the thymine of the A:T base pair at position 3 (GTAAAC) (Supplementary Fig. 3b) and to the N3 atom of the fourth adenine (GTAAAC). GTTTAC differs from GTTAAC at position 4, resulting in Arg203 making bidentate hydrogen bonds with the O2 atoms of the third and fourth thymines (GTTTAC) (Supplementary Fig. 3c). Thus, small adjustments in the configuration of the Arg203 side chain are able to accommodate all four variants of the GTWWAC recognition sequence.

DNA methylation

As noted above, the target adenine of DNA1 inserts into the catalytic cleft of molecule A and interacts with the conserved DPPY motif. Conversely, the target adenine of DNA2 enters the catalytic cleft of molecule B. The PPY sequence of the DPPY motif in molecules A and B runs along the Watson-Crick edge of the extrahelical adenine rather than the Hoogsteen edge as in the γ-class of amino MTases12. Considering DNA1 (and the same applies to DNA2), the extrahelical adenine stacks between Tyr62 and Thr216 and its N6 amino group makes hydrogen bonds with the Pro60 main chain carbonyl O and the Asp59 side chain OD1 atoms and a polar interaction with the side chain OH of Tyr68, and its N7 atom makes a hydrogen bond with Lys218. There are also putative weaker out-of-plane hydrogen bonds between its N1 atom and the main chain amide NH of Tyr62 and the side chain OH of Tyr68 (Figs. 3c and 4). Overall, this pattern of hydrogen bonding specifies an adenine in the catalytic cleft and positions its N6 amino group close to the methyl group of modeled SAM for methyl transfer by an in-line nucleophilic attack. To avoid catalysis and methyl transfer in our complex, we crystallized M.BceJIV in the presence of a SAM analog, sinefungin, which has a nontransferable amino group in place of the methyl group. Sinefungin is bound towards the C-terminal ends of strands β7 and β8 with the adenine ring stacked between Phe241 and Ile263, the sugar hydroxyls making hydrogen bonds with Glu262 OE1 and OE2 atoms, and the carboxyl end making hydrogen bonds with the main chain and side chain atoms of Ser244, main chain NH of Lys218 and OG1 atom of Thr246 (Fig. 4). The non-transferable amino group of sinefungin is located only 2.9 Å from the N6 atom of the extrahelical target adenine and is involved in polar interactions with the hydroxyl group of Tyr68 and with both the main chain and side chain atoms of Asp59.Fig. 4 Interactions of M.BceJIVΔ29 with sinefungin and the flipped adenine.

A close-up view of specific M.BceJIVΔ29-sinefungin interactions and residues shared between sinefungin and the flipped-out adenine. Sinefungin is tightly bound within the MTase domain via extensive hydrogen bonds (dashed lines) and hydrophobic contacts.

M.BceJIV binds two DNA sites in solution

To assess the stoichiometry of the M.BceJIV/DNA complex in solution, we used sedimentation velocity analytical ultracentrifugation experiments to observe the formation of the complex using the same 14-mer (containing the GTTAAC recognition sequence) used in co-crystallization. As shown in Fig. 5a, in the absence of DNA M.BceJIV sediments at ~ 4.30 S, corresponding to a molecular weight (MW) of 56.7 kDa, which is in excellent agreement with the expected MW of 58.3 kDa for an M.BceJIV dimer with two sinefungin molecules. In the presence of DNA (14mer), M.BceJIV sediments at ~ 5.29 S, corresponding to an MW of 77.0 kDa, which is in excellent agreement with the expected MW (75.3 kDa) for a two M.BceJIV/two DNA/two sinefungin complex. Together, the velocity sedimentation data supports the finding that M.BceJIV can simultaneously engage two DNA substrates.Fig. 5 M.BceJIV binds two DNA sites in solution and methylates them independently.

a Results from sedimentation velocity analysis showing the molecular weight distribution of M.BceJIVΔ29 in the absence of DNA (blue) and in the presence of DNA (red). The small peaks at the low end of the distribution are small contaminants, as confirmed by SDS-PAGE. The experiments were repeated in duplicates or triplicates, and the average molecular weight was reported. b Addition of a second DNA methylation site in trans (left) or in cis (right) does not assist the methylation of the first DNA site. In the trans experiment (left), HpaI digestion of 24 nM BamHI-linearized pUC18 containing a single HpaI site with increasing concentrations (0 nM to 50 nM) of a 19 mer DNA duplex in trans containing an M.BceJIV methylation motif and no HpaI site in the presence of 24 nM M.BceJIV and 20 μM SAM in a 50 s reaction. In the cis experiment (right), HpaI digestion of 24 nM BamHI-linearized pUC18 containing a single HpaI site (or both an HpaI site and another M.BceJIV methylation site in cis) with increasing reaction time (1 min to 5 min) in the presence of 24 nM or 48 nM M.BceJIV and 20 μM SAM. The experiments were repeated twice with similar results.

M.BceJIV methylates the two DNA sites independently

The finding that M.BceJIV can simultaneously bind two DNA substrates prompts the question of whether the binding and methylation of one DNA substrate facilitates the binding and methylation of the second DNA substrate. Many restriction enzymes are found to require two or more DNA sites for cleavage13–16 but whether a similar cooperation exists for a DNA MTase is unclear. To determine if methylation on a motif is dependent on the methylation of another motif, a set of experiments coupling the MTase activity of full-length M.BceJIV with the endonuclease activity of HpaI (NEB) was performed. HpaI is a restriction endonuclease that introduces a double-stranded break in the middle of the sequence 5’-GTTAAC-3’, which is a motif that contains an adenine (underlined) that gets methylated by M.BceJIV. We supplied the second motif in trans or cis and asked whether it leads to stimulation in methylation of the first site (see Supplementary Fig. 4). For these experiments, we constructed a plasmid carrying a single GTTAAC sequence that serves as a recognition site for both M.BceJIV (GTWWAC) and the restriction endonuclease HpaI (GTT▼AAC). The addition of HpaI to the linearized plasmid led to complete cleavage that could be visualized on an agarose gel with ethidium bromide. As expected, the addition of M.BceJIV and cofactor SAM to the mixture led to the inhibition of cleavage by HpaI. At appropriate concentrations of the enzymes and times for the reaction corresponding to ~ 50% inhibition of DNA cleavage, we tested whether the addition of an oligonucleotide in trans carrying the recognition sequence for M.BceJIV (GTAAAC), but not for HpaI, would stimulate methylation of the first site on the linearized plasmid (monitored as a reduction in cleavage by HpaI). The assay is analogous to that reported previously for the MmeI family of restriction enzymes which revealed stimulation of cleavage at one site when another is supplied in trans17. As shown in Fig. 5b, we observed no stimulation in methylation by M.BceJIV of the site on the plasmid when another was provided in trans. If anything, there is a slight inhibition in methylation of the first site when the second was provided in trans, presumably due to titration of the enzyme by the second site (Fig. 5b). Thus, even though the M.BceJIV dimer, from our structure, can simultaneously bind and methylate two recognition sequences, it appears to do so independently without cooperativity between the sites. To test whether this was also the case when the two sites were in cis, we constructed a plasmid carrying two recognition sequences, one for M.BceJIV/HpaI (site 1, GTT▼AAC) and another for just M.BceJIV (site 2, GTAAAC) (Supplementary Fig. 4) and monitored for HpaI cleavage of site 1 as a proxy for the degree of methylation. As shown in Fig. 5b, there is no stimulation of methylation of site 1 when the plasmid contains another M.BceJIV recognition sequence in cis. Thus, the M.BceJIV dimer can simultaneously bind and methylate two recognition sequences, but this occurs independently and without cooperativity between the sites.

Genomic distribution of M.BceJIV recognition sites

To understand the genome-wide distribution of the M.BceJIV recognition sites (5’-GTWWAC-3’), we conducted a genomic analysis of two B. cenocepacia strains, J2315 and K56-2, each with an approximate size of 8 Mb. Both strains comprise three chromosomes and one plasmid (Fig. 6a, b). Within strain J2315, we identified a total of 982 GTWWAC sites with a wide range of distances between each two neighboring sites (Fig. 6c). While most neighboring sites were about 7 kb from each other, some sites are as close as < 10 bp and as far as > 10 kb from their neighboring sites. The other strain K56-2, with 933 GTWWAC sites, exhibited a comparable pattern in the distribution of GTWWAC sites and distances between neighboring sites as strain J2315 (Fig. 6d). When we examined the relative distribution of GTWWAC sites between intergenic and intragenic regions, we found that 35.7% of the motif sites are in the intergenic regions of the J2315 genome, representing nearly 3-fold enrichment considering the size of intergenic regions in the genome (~ 12.5%). Hypothesizing that this enrichment in the intergenic region is likely due to the unusual AT-rich 4-mer (TWWA) in the GTWWAC motif, we performed a Markov chain motif frequency analysis8 and found that the observed enrichment of GTWWAC in the intergenic region is indeed largely driven by the AT-rich nature of intergenic regions compared to intragenic regions (Fig. 6e, f). These motif distribution analyses highlight that the GTWWAC methylation motif may mediate the competition between M.BceJIV and other DNA binding proteins, such as transcription factors, to facilitate epigenetic regulation3.Fig. 6 Distribution of GTWWAC motif sites across two strains of B. cenocepacia.

a, b Circos plot showing the genome-wide distribution of GTWWAC sites along the three chromosomes and a plasmid of B. cenocepacia J2315 and B. cenocepacia K56-2, respectively. c, d Density plot for the distribution of distances between each two neighboring GTWWAC sites in B. cenocepacia strains J2315 and K56-2. e, f Enrichment of GTWWAC in the intergenic regions (observed frequency vs. expected frequency) in the two strains.

Given that the structures of numerous DNA MTases are presently available, we searched the PDB database for MTases with high similarity. The enzyme M.RsrI from Rhodobacter sphaeroides displayed the highest degree of similarity, sharing 40.0% amino acid identity with the strains studied18. In addition, an amino acid identity of 36.2% was observed between M.BceJIV and M1.MboII from Moraxella bovis, the latter being an enzyme that methylates the 3’ adenine of GAAGA and usually forms a dimer when it is not bound to DNA19.

Binding and kinetic parameters

We measured the binding affinities of SAM, SAH, and sinefungin for M.BceJIV by isothermal titration calorimetry (ITC). From the ITC analysis, SAM and sinefungin bind M.BceJIV with similar affinities (KDs of ~ 8 μM), but SAH binds with approximately a two-fold weaker affinity (KD of ~ 17 μM) (Fig. 7a and Table 1). Energy-minimized docking structures of SAM and SAH at the crystallographic binding pockets of sinefungin in the M.BceJIV-DNA dimeric complex surrounded by an explicit water environment reveal differences in the interactions between the three ligands and the protein, particularly in regions where their chemical structures vary (Fig. 7b). Sinefungin is stabilized in part by an electrostatic interaction that its non-transferable, positively charged amino group establishes with Asp59, as well as hydrogen bonds formed with the main chain carbonyl oxygen atom, one of the side chain carboxylate oxygen atoms of Asp59, and the hydroxyl group of Tyr68. Similarly, SAM is stabilized by an electrostatic interaction between its positively charged sulfonium group and Asp59, along with possible unconventional C-H··O hydrogen bonds between its sulfonium methyl group and the main chain carbonyl oxygen atom, one of the side chain carboxylate oxygen atoms of Asp59, and the hydroxyl group of Tyr68. In addition, there may be possible sulfonium−π interactions with Tyr68. Notably, C-H··O bonds have been postulated in SAM-dependent methyltransferases20 due to the polarization of the methyl carbon within the positively charged sulfonium group. A recent study has demonstrated that this polarization also contributes to making sulfonium−π interactions stronger than sulfur−π and ammonium−π interactions, mostly because of the additional methyl−π interaction offered by the sulfonium group21. Unlike sinefungin and SAM, SAH is unable to participate in equivalent electrostatic, polar, and methyl−π interactions, which may account for its lower affinity.Fig. 7 ITC analysis of M.BceJIVΔ29 with SAM, SAH and sinefungin, and kinetics of M.BceJIV using a bioluminescence assay.

a ITC titration data for sinefungin (left), SAM (middle), and SAH (right) with M.BceJIVΔ29. The equilibrium dissociation constants (KD) were derived from the resulting binding isotherms. All the experiments were repeated in duplicates and the average value is reported. b Two-dimensional protein-ligand interaction diagrams generated using energy-minimized conformations of the crystal structure of M.BceJIVΔ29 bound to sinefungin (left) and predicted structures of SAM and SAH in M.BceJIVΔ29 (middle and right, respectively) embedded in an explicit water environment. The Ligand Interaction script in Maestro (Schrödinger Inc., http://www.schrodinger.com/) was used to generate these diagrams with default parameters. Among the protein residues within a 4 Å distance of the ligands, only those that interact with the ligands according to Maestro’s default parameters (see details in Supplementary Table 2) are displayed. c Kinetics of SAH byproduct formation by varying concentrations of the methyl donor SAM. A summary of kinetic parameters is also shown. Data are presented as mean values +/− SD from experiments performed in triplicates, and the source data are provided as a Source Data file.

Table 1 Thermodynamic parameters of M.BceJI binding to sinefungin, SAM, and SAH

Ligand	KD (µM)	Stoichiometry (N)	ΔG (Kcal/mol)	ΔH (Kcal/mol)	-TΔS (Kcal/mol)	
Sinefungin	8.35 ± 0.39	0.84 ± 0.01	− 6.94 ± 0.02	− 10.8 ± 0.3	0.09 ± 0.16	
SAM	8.26 ± 0.45	0.98 ± 0.02	− 6.94 ± 0.03	− 10.8 ± 0.5	0.25 ± 0.03	
SAH	17 ± 2	0.80 ± 0.04	− 6.52 ± 0.06	− 16 ± 1	0.78 ± 0.38	

We next measured the M.BceJIV kinetic parameters by measuring concentrations of the reaction product SAH under varying concentrations of SAM. From the kinetic analysis, M.BceJIV has Km and kcat values of ~ 5.9 μM and ~ 1.5 min−1, respectively (Fig. 7c). The Km value is approximately similar to the KD value for SAM determined from ITC analysis. The kcat value is at the lower end of values reported for other orphan DNA MTases, namely E. coli Dam (~ 0.14 min−1)22, C. difficile CamA (~ 5.4 min−1)5, and C. crescentus CcrM (~ 5.2 min−1)23. Like M.BceJIV, CcrM is also a dimer and belongs to the β class of amino MTases, but unlike M.BceJIV, it engages only a single DNA substrate as a dimer24. Thus, even though the M.BceJIV dimer engages two DNA substrates for methylation, it does not lead to a faster catalytic turnover of the substrate than CcrM.

Discussion

We present here the crystal structures of the M.BceJIV/DNA/sinefungin ternary complex. M.BceJIV is only the third β class of amino MTases to be visualized with DNA. The first one, EcoP15I, revealed dimerization as the defining feature of this class of MTases and uncovered a remarkable division of labor, where one MTase subunit recognizes the DNA, while the other MTase subunit methylates the target adenine25. The second, cell-cycle regulated CcrM, also revealed a dimer, wherein the two subunits cooperate in pulling apart the DNA strands over the recognition sequence24. In both the EcoP15I and CcrM complexes, the MTase dimer binds a single DNA duplex. M.BceJIV is also a dimer, but it engages not one, but two DNA duplexes, wherein each subunit contributes to the binding and recognition of both DNA duplexes. Thus, even though M.BceJIV, EcoP15I and CcrM belong to the same β class of amino MTases and are homodimers, there is surprising diversity in how they distribute labor between the subunits for DNA binding and methylation.

The ability of M.BceJIV to engage two recognition sequences posits an ability to co-methylate two DNA sites at the same time, whether they occur in trans or cis. The cis sites on a bacterial chromosome could be potentially far apart with the DNA in between them looped out when they bind the M.BceJIV dimer. To test whether co-methylation is cooperative, we monitored the extent of methylation at one DNA site when another is supplied in trans or cis. From these experiments, the methylation on one site does not stimulate the methylation of the second site, suggesting that co-methylation on two DNA sites occurs independently without cooperativity. From our bioinformatic analysis of the B. cenocepacia genome, the M.BceJIV recognition sequences are distributed with a wide range of distance between each two neighboring sites. While most neighboring sites were about 7 kb from each other, some sites are as close as a few base pairs and as far as > 10 kb from their neighboring sites. Intriguingly, the sites are significantly enriched in intergenic regions, a reflection perhaps of the regulatory role of M.BceJIV in gene transcription.

Bacterial DNA MTases are commonly part of the R-M systems for defense against phages2. As an orphan MTase (unassociated with a restriction enzyme), M.BceJIV joins a growing number of bacterial MTases with putative roles outside of phage defense4,7. A recent study in B. cenocepacia strains J2351 and K56-2 showed a role for M.BceJIV in the epigenetic regulation of gene expression related to cellular motility, making M.BceJIV a viable therapeutic target for limiting B. cenocepacia infections10. A potential set of therapeutics is small molecules that compete with the binding of the cofactor SAM to M.BceJIV. Indeed, many SAM-mimetic and non-SAM mimetic compounds have been developed to inhibit protein MTases in the cancer field26,27, and more recently against the SARS-CoV-2 MTases during the COVID-19 pandemic28,29. It would be interesting to test some of these for inhibition of M.BceJIV as starting lead compounds. The structure of M.BceJIV provides a basis for optimizing these lead compounds for increased potency and selectivity through iterative cycles of crystallography, chemical synthesis, and assays for B. cenocepacia’s motility. Interestingly, B. cenocepacia wild-type cells treated with sinefungin are less motile (to a similar extent as an M.BceJIV deletion mutant), consistent with the inhibition of M.BceJIV in a cellular context10. One issue with sinefungin as a potential therapeutic is that it is a pan MTase inhibitor with potential off-target effects, including the inhibition of host MTases in any treatment regimen. However, it is noteworthy from our studies that sinefungin binds M.BceJIV with a similar affinity as SAM (KDs of ~ 8 μM) due to favorable interactions between its non-transferable positively charged amino group and Asp59 and Tyr68; positing sinefungin as a good starting scaffold for the design of SAM-mimetics selective for M.BceJIV for inhibition of B. cenocepacia’s motility. Altogether, inhibition of bacterial DNA MTases for controlling bacterial infections has gained traction over the past few years5,6,8. A notable example is the development of selective and potent inhibitors (KD of ~ 0.2 μM) for the DNA adenine MTase CamA for controlling C. difficile’s sporulation and biofilm formation9.

In summary, we present here the crystal structures of the M.BceJIV/DNA/sinefungin ternary complex. The structures reveal a mode of DNA binding wherein each M.BceJIV monomer contributes to the recognition of two DNA sequences. Given the role of M.BceJIV in regulating B. cenocepacia’s cellular motility, it is a viable therapeutic target, and the structure presented here will be invaluable in developing selective and potent inhibitors of M.BceJIV for limiting B. cenocepacia infections.

Methods

Expression and purification

For the expression of full-length N-terminal His tagged M.BceJIV (Uniprot Accession number B4EFK1), the codon-optimized gene was cloned into a pET28-smt3 vector. For crystallization, a construct of M.BceJIV without the first 29 N-terminal residues (M.BceJIVΔ29) was cloned in the pET28-smt3 vector with N-terminal 6xHis tag. The plasmids were transformed into E. coli NiCo21(DE3) cells (NEB) and the cells were grown in LB media supplemented with 50 µg/mL Kanamycin at 37 °C until OD600 reached 0.8. Protein expression was induced with 0.6 mM isopropyl β-D-1-thiogalactopyranoside and cultures were incubated for 17 h at 18 °C. The cell pellets from 4 L were resuspended in 150 mL binding buffer (50 mM Potassium phosphate pH 8.0, 500 mM Sodium chloride (NaCl), 5% glycerol, 0.01% triton X-100, 5 mM β-mercaptoethanol, 30 mM Imidazole and 10 μM Zinc chloride) containing 1 mM Phenylmethylsulfonyl fluoride and protease inhibitors. Cells were sonicated on ice for 10 min with 15 s ON, and 2 min OFF, and cell debris was removed by centrifugation at 38,828 rcf. The supernatant was filtered and incubated with 5 mL of Ni-NTA beads (Thermo Scientific) for 1 h. The beads were then washed with 100 mL binding buffer followed by 100 mL high salt buffer (binding buffer with an additional 500 mM NaCl) and 30 mL binding buffer. The protein was then eluted with elution buffer (binding buffer with 500 mM imidazole), and the His tag was cleaved using Ulp1-protease by dialyzing overnight at 4 °C against 1 L binding buffer with 100 mM NaCl. To remove the cleaved His-tag, the protein was passed through a 5 mL HisTrap HP column (GE Healthcare), and the cleaved protein was collected in the flow through. The protein was further purified using a 5 mL HiTrap Heparin HP column (GE Healthcare) and then eluted with a linear gradient of 0.1–0.5 M NaCl. The peak elution fractions were pooled and concentrated to 2 mL using an Amicon Ultra 10 K MWCO centrifugal filter at 4 °C and loaded into a HiLoad Superdex 200 16/60 column (GE Healthcare) equilibrated with 20 mM HEPES pH 8.0, 300 mM NaCl, 5% glycerol and 0.5 mM tris(2-carboxyethyl)phosphine. The purified protein was concentrated to > 35 mg/mL, aliquoted, and stored at − 80 °C.

Crystallization and structure determination

For crystallization experiments, the ternary complex was reconstituted by mixing 20 mg/mL M.BceJIVΔ29, 2.5 mM sinefungin, and 0.83 mM DNA duplex (5′-TTGTTAACTAGCCA-3’ and 3′-ACAATTGATCGGTA-5′) in size exclusion buffer. The mix was incubated for at least 1 h in ice followed by centrifugation at 15,900 rcf at 4 °C for 10 min to remove any precipitate. Initial crystallization screening was done with commercially available screens using an Oryx Nano (Douglas Instruments) in a sitting-drop format with 0.25:0.25 µL protein-reservoir ratio at 17 °C. The initial crystals were obtained within 2 d in solution containing 0.10% w/v n-Octyl-β-D-glucoside, 0.1 M Sodium citrate tribasic dihydrate pH 5.5, and 22% w/v Polyethylene glycol 3350. The crystals were further optimized by varying the pH of the crystallization buffer, NaCl concentration in the sample mix, and percent of precipitant in a hanging-drop format using 1:1 and 1:1.25 µL protein-reservoir ratios. Crystals were cryoprotected in a stepwise manner with a reservoir solution containing 5–25% sucrose and flash-frozen in liquid nitrogen after 1–5 min incubation. Crystallization with oligonucleotides containing recognition sequence variants GTATAC, GTAAAC, and GTTTAC was conducted in the same manner.

X-ray diffraction data were collected under cryogenic conditions at the National Synchrotron Light Source II 17-ID-1 and 17-ID-2 beamlines at the Brookhaven National Laboratory. The diffraction data were processed using autoPROC and STARANISO (Global Phasing Ltd.). The structure with the recognition sequence variant GTTAAC was solved first by molecular replacement (MR) using Phaser-MR with the AlphaFold30 model of M.BceJIV as the search model, and to model the DNA molecules the PDB entry 6PBD was utilized as a reference. Subsequent iterative manual binding was performed with Coot31 and refinement with Phenix Refine32. A ligand restraint file for sinefungin was generated with eLBOW33 from the PHENIX suite. The structures with the recognition sequence variants GTATAC, GTAAAC, and GTTTAC were solved using the structure with GTTAAC (above) as an MR model. PyMOL (Schrödinger LLC) was used to prepare all the structure figures.

Analytical ultracentrifugation sedimentation velocity

The sample mix containing 70 µM M.BceJIVΔ29 and three times excess sinefungin was dialyzed overnight at 4 °C against buffer containing 20 mM HEPES pH 8.0, 170 mM NaCl, 2.5% glycerol, and 0.5 mM TCEP. The sample mix containing M.BceJIVΔ29 in complex with sinefungin and 1.2 times excess 14-mer DNA duplex (5′-TTGTTAACTAGCCA-3’ and 3′-ACAATTGATCGGTA-5′) was dialyzed overnight at 4 °C against buffer containing 20 mM HEPES pH 8.0, 50 mM NaCl, 2.5% glycerol, and 0.5 mM TCEP. The samples were incubated for at least 1 hr on ice before dialysis.

Analytical ultracentrifugation was performed in a Beckman XL-A/I analytical ultracentrifuge (Beckman-Coulter, Palo Alto CA, USA) in the sedimentation velocity mode, using two-cell centerpieces with tapered sides, 12 mm optical path length, and sapphire windows and at a speed of 116,444 rcf in an An-50 Ti rotor. Data were collected by interference optics at 675 nm monochromatic light, and scans were taken every two minutes. Buffer density and viscosity, as well as protein v-bar, were calculated using the program SednTerp (Alliance Protein Laboratories, Corte Cancion, Thousand Oaks, CA, USA). Raw data was evaluated and analyzed, and the molecular weights calculated using the program SedFit obtained from the National Institutes of Health (www.nibib.nih.gov). The experiments were repeated in duplicates or triplicates, and the average molecular weight was reported.

Methylation-endonuclease coupled assays

For methylation-endonuclease coupled assays, pUC18 plasmids were linearized by digestion with BamHI prior to the experiments. For the in trans experiments, increasing concentrations (0 nM to 50 nM) of a 19-mer DNA duplex (5’-GACGTGTAAACATGCGCGTC-3’ and 3’- GCTGCACATTTGTACGCGCA-5’) containing an M.BceJIV methylation motif and no HpaI endonuclease site was added in trans when indicated, and the mix was further incubated for 30 min at room temperature in reaction buffer (20 mM Tris pH 8.0, 50 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 3 mM magnesium chloride, 1 mM dithiothreitol and 0.1 mg/ml Bovine Serum Albumin). For the in cis experiments, methylation-endonuclease activity was assayed in 20 µL reaction mixes by incubating 24 nM or 48 nM of full-length M.BceJIV with 24 nM of pUC18 plasmids containing an HpaI site only (or both an HpaI site and another M.BceJIV recognition site in cis) for 30 min at room temperature in reaction buffer. For all the experiments, the methyltransferase reactions were started by adding 20 μM SAM and proceeding for 50 s (or increasing reaction times from 1 to 5 min) before adding HpaI in excess and incubating the mixes for at least 1 h at 37 °C in a final reaction volume of 20 µL. Reaction products were analyzed by electrophoresis alongside a 1-kb DNA ladder in 1% SeaKem LE agarose gels prepared and run in 1X TAE with ethidium bromide. All the experiments were repeated twice.

Methylation motif distribution analysis

We obtained the genome assemblies for the two B. cenocepacia strains from the NCBI’s Genbank database, under accession numbers AM747720.1 - AM747723.1 (for strain J2315) and Z_CP053300.1 - Z_CP053303.1 (for strain K56-2). The GTWWAC sites were identified by employing the seqkit program34 (v.0.15), and their distribution was visualized via a circos plot, which was created using the circlize package in R. In order to identify the homolog of M.BceJIV, we conducted a BLASTP search against the PDB database, using the M.BceJIV amino acid sequence as the query. The expected frequency of the methylation motif was estimated using Markov chain motif distribution analysis as previously described8. That is, previously we computed the exceptionality for a k-mers motif by comparing observed and expected counts under Markov models. Here, the expected frequency of the GTWWAC motif is calculated as f(TWWAC) * f(GTWWA) / f(TWWA), where f() represents the frequency of a motif in a specific genome.

Isothermal titration calorimetry assays

The isothermal titration calorimetry experiments were performed on a MicroCal PEAQ-ITC (Malvern Panalytical). The ligands (400 µM) were loaded in the syringe and titrated into 40 µM M.BceJIVΔ29 in the cell. Titrations were performed at 25 °C with the standard 10 μcal/s reference power at 600 rpm. The titrations consisted of an initial injection of 0.4 µL (excluded from analysis) followed by 15 injections of 2.5 µL ligand at a rate of 0.5 µL/s at 180 s time intervals. Care was taken to ensure buffer match for the ligand and M.BceJIVΔ29 to eliminate heat from buffer mismatch. Data were fit into a single binding site model and analyzed using the MicroCal PEAQ-ITC Analysis software v1.41. All the experiments were repeated in duplicates, and the average value was reported.

Docking of SAM and SAH to the sinefungin’s binding sites in the crystal structure of the M.BceJIV-DNA dimeric complex

The ligands and the protein-DNA dimeric complexes were prepared for docking using the default protocols in LigPrep and the Protein Preparation Wizard from the Schrödinger 2020-4 suite35, respectively. Briefly, a docking grid with edge lengths of 12 Å × 13 Å × 17 Å was generated and centered on the center of mass of sinefungin, using the Schrodinger′s Receptor Grid Generation module35. Both SAM and SAH were docked using Glide with the Standard Precision (SP) scoring function35 after validating the protocol with sinefungin as a control.

Energy minimization

Energy minimizations were carried out using GROMACS (version 2021.4)36 to assess the stability of the crystallographic binding pose of sinefungin and the predicted binding poses of SAM and SAH in the same binding pocket of the protein-DNA complexes embedded in an explicit water environment. Specifically, all three ligand-bound protein-DNA dimeric complexes were embedded in a cubic box with a 10.8 nm edge length in each dimension filled with TIP3P water molecules and 0.15 M NaCl to neutralize the systems, using the Solution Builder tool in CHARMM-GUI37. The CHARMM36m force field38 was used to parameterize the protein, DNA, and solvent in the systems. While the parameters for SAM were present in the July 2022 release of the force field, the parameters for SAH and sinefungin had to be generated using the CHARMM General Force Field (CGenFF) webserver (https://cgenff.silcsbio.com/)39. After performing energy minimization cycles for 1,000,000 steps with positional restraints on each of the ligand-bound protein-DNA systems, additional energy minimization cycles in which the systems were unrestrained were conducted for 1,000,000 steps.

Methylation kinetics assays

The SAM-dependent DNA methylation by full-length M.BceJIV was measured using Promega MTase-GloTM luminescence assay40 where the SAH product is converted to ADP and then ATP. The 20 µL reaction mix contained 20 nM M.BceJIV, increasing concentrations (0 µM to 23 µM) of SAM, and 20 µM DNA duplex (5’-GACGTGTTAACATGCGCGTC-3’ and 3’-GCTGCACAATTGTACGCGCA-5’) in the same buffer composition described in the methylation-endonuclease assays section. DNA and SAM were preincubated for 5 min before starting the reaction. The SAH calibration curve was generated using 0, 0.125, 0.25, and 0.5 µM SAH (Supplementary Fig. 5). The reactions were performed at room temperature and terminated after 10 min by adding 5 µL of 0.5% trifluoroacetic acid. Five microliters of 6X MTase-GloTM reagent were added to the mix and incubated for 30 min before adding 30 µL of MTase-GloTM detection solution and incubating for an additional 30 min. Luminescence was measured with a TECAN Infinite 200 Pro Microplate Reader. The data were analyzed using GraphPad Prism v9. All the experiments were repeated in triplicates.

Reporting summary

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

Supplementary information

Supplementary Information

Peer Review File

Reporting Summary

Source data

Source Data

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-52130-x.

Acknowledgements

This work was funded by grants R35-GM131780 (to A.K.A) and R35GM139655 (to G.F.) from the National Institutes of Health (NIH). We thank the staff at the National Synchrotron Light Source II (NSLS-II) beamlines 17-ID-1 and 17-ID-2 for facilitating X-ray data collection. NSLS-II is a United States Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. The Center for BioMolecular Structure (CBMS) at NSLS-II is primarily supported by the NIH, National Institute of General Medical Sciences (NIGMS) through a Center Core P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1605010). Computational resources needed for this work were provided in part by Scientific Computing at the Icahn School of Medicine at Mount Sinai, the Clinical and Translational Science Awards (CTSA) grant UL1TR004419 from the National Center for Advancing Translational Sciences, and the Office of Research Infrastructure of the National Institutes of Health under award number S10OD026880.

Author contributions

A.K.A, G.F, M.F. and J.K. designed the study; R.Q.-F. performed the crystallographic, biophysical, and biochemical studies; J.K. assisted and guided the crystallographic, biophysical, and biochemical studies; M.N. performed the methylation motif distribution analysis; R.G. and L.S.-E. performed and analyzed the energy minimization and docking studies; G.A. performed the analytical ultracentrifugation sedimentation velocity experiments with R.Q.-F. assistance; O.R. assisted in kinetic and crystallographic analysis; G.F. guided the methylation motif distribution analysis; M.F. guided the analysis of the energy minimization and docking studies; L.S guided the sedimentation velocity experiments; A.K.A. guided the overall project; A.K.A., M.F., G.F., J.K. and R.Q.-F. wrote the manuscript with input from all the authors.

Peer review

Peer review information

Nature Communications thanks Evzen Boura, and Richard Roberts for their contribution to the peer review of this work. A peer review file is available.

Data availability

The structure factors and coordinate files for crystal structures of the M.BceJI/DNA/sinefungin ternary complexes with GTTAAC, GTATAC, GTAAAC, and GTTTAC recognition motifs have been deposited in the Protein Data Bank (PDB) under the accession codes 8URK, 9C3S, 9C3T, and 9C3U, respectively. The following published structure was used in this work: 6PBD. The raw data for the Kinetic assay (for Fig. 7c) are available as source data files accompanying this manuscript. Source data are provided in this paper.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Richard Quintana-Feliciano, Jithesh Kottur.
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