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Novel structural features drive DNA binding properties of Cmr, a CRP family protein in TB complex mycobacteria

Abstract Mycobacterium tuberculosis (Mtb) encodes two CRP/FNR family transcription factors (TF) that contribute to virulence, Cmr (Rv1675c) and CRPMt (Rv3676). Prior studies identified distinct chromosomal binding profiles for each TF despite their recognizing overlapping DNA motifs. The present study shows that Cmr binding specificity is determined by discriminator nucleotides at motif positions 4 and 13. X-ray crystallography and targeted mutational analyses identified an arginine-rich loop that expands Cmr’s DNA interactions beyond the classical helix-turn-helix contacts common to all CRP/FNR family members and facilitates binding to imperfect DNA sequences. Cmr binding to DNA results in a pronounced asymmetric bending of the DNA and its high level of cooperativity is consistent with DNA-facilitated dimerization. A unique N-terminal extension inserts between the DNA binding and dimerization domains, partially occluding the site where the canonical cAMP binding pocket is found. However, an unstructured region of this N-terminus may help modulate Cmr activity in response to cellular signals. Cmr’s multiple levels of DNA interaction likely enhance its ability to integrate diverse gene regulatory signals, while its novel structural features establish Cmr as an atypical CRP/FNR family member.

INTRODUCTION
Mycobacterium tuberculosis (Mtb) is the etiologic agent of tuberculosis (TB), which causes high levels of morbidity and mortality worldwide (1). Mtb adapts to changing conditions during infection by modulating its gene expression, and the second messenger cyclic AMP (cAMP) plays a key role in Mtb's gene regulatory response to host-associated conditions (2,3). CRP Mt (Rv3676) and Cmr (Rv1675c) are cAMP-associated transcription factors (TFs) in Mtb, and both are classified as CRP/FNR family TFs (4).
The CRP/FNR family of TFs is named after the two earliest discovered members of the group, cAMP receptor protein (CRP) and fumarate and nitrate reductase (Fnr) in Escherichia coli (5,6). (E. coli CRP is referred to herein as CRP Ec for clarity, and is also called CAP, for catabolite activator protein). All CRP/FNR family TFs are characterized by the presence of an N-terminal ␤-barrel/cyclic nucleotide binding domain, and a C-terminal helix-turnhelix (HTH) DNA binding domain (6,7). Advancement in genome analysis and phylogenetic methods has resulted in increased detection of CRP/FNR type TFs (6,8). A study classified ∼1500 CRP/FNR type regulators into 12 representative groups in 2012 (8). CRP/FNR family TFs recognize and respond to a variety of environmental signals, including cAMP, carbon monoxide, 2-oxoglutarate and cellular redox state (6,7,(9)(10)(11)(12)(13). These TFs are also known to regulate diverse cellular processes such as catabolite repression, nitrogen fixation, metabolism, respiration, aromatic ring degradation and expression of virulence factors (6,(14)(15)(16)(17)(18).
CRP Mt is the closest Mtb ortholog of CRP Ec and is better studied than Cmr (19)(20)(21)(22)(23). Both Cmr and CRP Mt exhibit widespread genomic binding associated with the control of large putative regulons (24)(25)(26), and deletion of either gene attenuates Mtb virulence in a murine model (21,27). CRP Mt regulates serine metabolism and is required for wild-type (WT) levels of growth in liquid culture and within macrophages (20,21,28). In contrast, Cmr coregulates genes within the dormancy associated DosR regulon and contributes to nitric oxide sensitivity (26,27) but cmr-deleted mutants are not defective for in vitro growth in nutrient-rich media (29).
Cmr was first identified as the regulator of a group of cAMP-and macrophage-responsive genes in Mtb (29). The genome-wide binding sites of Cmr have now been identified (26). Despite recognizing a 16-bp motif (NGTC/G-N 8 -G/CACN) that fully overlaps that of CRP Mt , there is minimal overlap between the genomic binding targets of the two TFs (24)(25)(26)29). Cmr binds ∼368 genomic loci in TB complex mycobacteria while CRP Mt binds ∼2000 (24,26). The N-terminal region of Cmr has a putative nucleotide-sensing domain (4). cAMP affects Cmr's DNA binding enrichment at a subset of sites in vivo, particularly in the vicinity of genes associated with the hypoxia-inducible DosR (DevR) regulon in Mtb (26). Addition of cAMP also causes this subset of DNA binding sites to migrate anomalously when complexed with Cmr during electromobility shift assays (EMSA) (26). Nonetheless, direct binding of cAMP to Cmr has not been demonstrated and the basis for differences in DNA and cAMP binding properties between Cmr and CRP Mt is not known.
CRP Ec and CRP Mt have been extensively studied, and serve as prototypes for understanding the CRP/FNR family of DNA binding proteins. CRP Mt and CRP Ec recognize and bind to the DNA motifs (C/TGTG/CA/G-N 6 -T/CC/GACG/A) and (TGTGA-N 6 -TCACA), respectively via their C-terminal HTH domains (20)(21)(22)24,25,30). The differences between CRP Ec and CRP Mt become more apparent when comparing their functional dependence on cAMP binding. CRP Ec cannot bind DNA in the absence of cAMP, which causes elaborate conformational changes that position the HTH domains for DNA binding (30,31). In contrast, CRP Mt shows specific DNA binding at most sites even in the absence of cAMP (19,22), although direct binding of cAMP to CRP Mt enhances its DNA binding affinity ∼2-fold (20,21).
In this study, we determined a high-resolution crystal structure of Cmr, and analyzed the key features of this protein using mutational and molecular methods. Our study identifies novel structural regions in Cmr that play vital roles in determining the specificity of DNA recognition and binding. We show that accessory DNA contact regions that are not present in CRP Mt influence the ability of Cmr to distinguish between sites with single nucleotide differences. Additionally, Cmr binding to DNA is cooperative and appears to bend the DNA asymmetrically. Along with its unique DNA binding features, the intriguing lack of a cAMP-binding pocket differentiates Cmr from the CRP prototypes of this family.

Recombinant DNA construction (For X-ray crystallization)
DNA fragments encoding Cmr 1-244 and Cmr 13-244 were amplified by polymerase chain reaction (PCR) from template plasmid pMBC370, containing the cmr open reading frame cloned downstream of the N-terminal His6x tag in the pet28a+ vector backbone, and inserted into a modified pMCSG9 vector (32) using ligation-independent cloning (33) to create pMCSG9 Cmr1 and pMCSG9 Cmr13. This fused a decahistidine-tagged maltose-binding-protein (His 10 MBP) at the N-terminal of Cmr that could be cleaved using tobacco etch virus (TEV) protease.

Protein expression and purification (For X-ray crystallization)
Expression vectors pMCSG9 Cmr1 and pMCSG9 Cmr13 were transformed into E. coli BL21(DE3) cells. Using the autoinduction method (34), initial cultures in 100 mM phosphate non-inducing minimal media (PAG) containing 100 g/ml carbenicillin were grown overnight at 37 • C and then subsequently inoculated into autoinducing PA-5052 or PASM-5052 media (100 g/ml carbenicillin added in both) for expression of either unlabeled native protein or selenomethionyl(SeMet)-protein for structure determination, respectively. Fusion proteins were expressed overnight at 30 • C and purified via two-step nickel-affinity/gel filtration chromatography using a previously described method (35).

Crystallization and data collection
Crystals from which structures were determined did not require optimization and were grown using the sitting-drop vapor diffusion method directly from initial sparse matrix screening in which 0.2 L of protein was mixed with an equal volume of crystallization buffer from commercial JCSG Core Suite I, II, III, and IV screens (Qiagen). At room temperature, Cmr 1-244 crystallized in a condition containing 20% (wt/vol) polyethylene glycol (PEG) 3350 and 0.2 M potassium sulphate, and Cmr 13-244 crystallized in a condition containing 1 M di-ammonium hydrogen phosphate, 0.1 M imidazole pH 8.0 and 0.2 mM NaCl. Crystals were flash frozen in liquid nitrogen after briefly soaking in a cryoprotectant solution containing the crystallization buffer supplemented with 18-20% (vol/vol) ethylene glycol. A dataset from a single crystal of each Cmr 1-244 and Cmr 13-244 was collected at the X4C beamline, tuned to the Se K-edge, at the National Synchrotron Light Source of Brookhaven National Laboratory. For Cmr 13-244, both a Nucleic Acids Research, 2018, Vol. 46, No. 1 405 high-angle and low-angle dataset was collected and subsequently combined.

Structure determination and refinement
X-ray diffraction data was indexed and merged using HKL2000 (6) and CCP4 (37). Phases for the structure of Cmr 13-244 were calculated by single-wavelength anomalous dispersion (SAD) (38) using the AutoSol script in Phenix (39) and yielded interpretable density-modified electron density maps in space group P2 1 2 1 2. ARP/wARP 7.5 (40) of the CCP4 package was used to build the initial model and COOT (41) and Phenix were used for final manual model building and refinement. The structure of Cmr 1-244 was solved using Cmr 13-244 as a molecular replacement search model using Phaser (42). Refinement of Cmr 1-244 was performed in a similar manner as Cmr 13-244. For both structures, a random 5% of reflections were used as the cross-validation set. Model validation statistics were calculated using Molprobity (43).

Motif analysis
The 30-nt sequences flanking the center of Cmr or CRP Mt binding sites (total length of sequences = 60 nt) were extracted from the H37Rv genome and used as input sequences for MEME motif analysis (44) (http://meme-suite. org/tools/meme). The 359 Cmr binding sites, and 1000 CRP Mt binding sites were considered for this analysis. The program was not restricted to search for palindromic motifs.

Cloning, expression and purification of proteins (for in vitro assays)
Recombinant His-tagged cmr (full length) and the HTH+ and HTH− truncated cmr were PCR amplified from M. tuberculosis H37Rv DNA template, using primers listed in Supplementary Table. S1. The amplified DNA products were cloned into pET28a+ (Novagen) between EcoRI and HindIII restriction sites to generate pMBC370, pMBC787 and pMBC788 respectively, all with a N-terminal His6x tag. C-terminal His6x tagged Cmr (pMBC1761) was expressed by cloning cmr ORF (open reading frame) into the NcoI and XhoI sites in the pET28a+ vector. Mutant proteins CmrR28A, CmrR35AR39A, Cmr-Arg loop * and CmrN 12 were constructed using sequence overlap extension primers listed in Supplementary Table. S1, and cloned into the NcoI and XhoI sites in pet28a+ vector to create strains pMBC1781, pMBC1782, pMBC1783 and pMBC1916 respectively. The plasmids were sequence verified, and maintained in E. coli BL21 (DE3) strain.
Bacterial cultures were grown to OD 600nm 0.4-0.6, and the expression of full length Cmr was induced with 1 mM isopropyl-␤-thiogalactopyranoside (IPTG) for 3 h at 21 • C. The expression of the truncated proteins was induced overnight at 15 • C with 1 mM IPTG. Protein expression was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis using anti-His monoclonal antibody (Clontech).
Protein purification was carried out as described previously (26). For purification, a 500-ml culture was pelleted, washed with 1 mM Tris-HCl, and resuspended in 12.5 ml lysis buffer containing 50 mM Tris-HCl (pH 8.0), 0.02% glycerol, 1 mM dithiothreitol and 1% protease inhibitor cocktail (Sigma). Bacteria were lysed by three freeze-thaw cycles and sonication for 5 min (at 4 • C), followed by three additional freeze-thaw cycles. The lysate was cleared by centrifugation at 13 000 rpm for 15 min at 4 • C. The His-tagged proteins were purified using HisTrap affinity column (GE Lifesciences) per the manufacturer's instructions, and the eluted protein was dialyzed against phosphate-buffered saline with 10% glycerol. Protein concentration was measured with a NanoOrange Protein Quantitation Kit (Molecular Probes) and diluted to 0.3 mg/ml before being stored in aliquots at −70 • C.

Cross-linking of Cmr by glutaraldehyde
Cross-linking of purified His-tagged Cmr was performed as previously described (19). His-Cmr was diluted to 310 nM in cross-linking buffer (50 mM sodium phosphate, pH 7.4, 20% glycerol, 5 mM MgCl 2 ), and was then incubated with glutaraldehyde at a final concentration of 7 mM for 1 h at room temperature. The reaction was quenched by the addition of SDS-PAGE sample buffer, and 8 l of cross-linking reaction was separated on a 12% SDS-PAGE gel. The protein was transferred onto a PVDF membrane and visualized by Western blot with anti-Cmr serum.

Electrophoretic mobility shift assays (EMSA)
PCR forward primers were labeled with [␥ -33 P]-ATP (MP Biomedicals or Perkin Elmer) using T4 DNA polynucleotide kinase (New England Biolabs). DNA probes were generated using labeled forward primer and unlabeled reverse primer in PCR reaction. About 0.05 pmol DNA probe was used in each 10 l binding reaction mixture. Briefly, 0.3 M His6x-Cmr (C-terminal tagged) and DNA probes were incubated at room temperature for 30 min in DNA binding buffer [10 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM EDTA, 50 g/ml bovine serum albumin, 1 mM dithiothreitol, 0.05% non-ionic P-40 detergent, 20 g/ml poly(dI-dC) and 10% glycerol], with or without 100M cAMP. Samples were loaded on a 6, 8 or 12 non-denaturing polyacrylamide gel, depending on the size of the DNA probe, and run for 2-3 h at 14 V/cm in 0.5× Tris-borate-EDTA buffer at 4 • C. Gels were transferred to Whatman paper, vacuum-dried, exposed overnight on a phosphor screen, scanned with Storm 860 PhosporImager (Molecular Dynamics), and analyzed with ImageQuant software (Molecular Dynamics).
To estimate the DNA binding affinity (K d ) of the proteins, EMSA experiments were performed by titrating increasing amounts of purified proteins into the DNA binding reaction, while keeping the concentration of radiolabeled DNA constant. The fraction bound in each lane was quantified using densitometry (ImageQuant, Molecular Dynamics). K d was calculated using nonlinear-regression analysis and curve fitting using 'Specific binding-One site with Hill slope' model in GraphPad Prism software. K d and Hill coefficient (h) were calculated using the formula where, 'Y' denotes specific fraction bound, 'B max ' denotes maximum specific binding, 'X' denotes ligand concentration, 'K d ' denotes binding affinity and 'h' denotes Hill coefficient.

Comparison of genome-wide binding profiles of Cmr and CRP Mt
Genome-wide binding sites for Cmr and CRP Mt were recently identified in TB complex mycobacteria using ChIPseq (24)(25)(26). Their genomic binding profiles were distinct, with CRP Mt binding nearly five times as many sites as Cmr bound (2000 versus 368, respectively) (24,26). However, MEME motif analysis (44) of the binding sites showed that the core 16-bp DNA binding motif recognized by Cmr (NGTC/G-N 8 -G/CACN) includes that of CRP Mt (C/TGTG/CA/G-N 6 -T/CC/GACG/A) (Supplementary Figure S1a) (20,24,26). Mutational analyses showed complete loss of in vitro DNA binding by either TF upon individually mutating any of the four critical nucleotide positions (2G, 3T, 14A and 15C), common to their binding sites (22,26). Nucleotide positions 4G and 13C were previously shown to also be critical for CRP Mt binding in vitro (20,23). Therefore, it was surprising that Cmr bound fewer genomic sites than CRP Mt despite binding a DNA motif that appears to be less specific than the CRP Mt binding motif. We compared the genomic DNA binding profiles of Cmr and CRP Mt by searching within published ChIP-seq datasets for the presence of CRP Mt binding sites within 500 bp of the center of each Cmr binding site (24,26). A total of 189 out of the 359 (52.6%) Cmr binding sites considered in this analysis had at least one CRP Mt binding site within 500 bp ( Figure 1A), and 95 (26.4%) Cmr binding sites were within 50 bp of a CRP Mt binding site ( Figure 1B). While some of these TF binding sites directly overlap, the majority do not. A total of 266 of 1700 (15.7%) CRP Mt binding sites considered in this analysis were within 500 bp, while 107 (6.3%) mapped within 50-bp of a Cmr binding site (Figure 1C). Thus, despite recognizing seemingly identical DNA binding motifs, Cmr and CRP Mt bind very distinct sites in the Mtb genome. While these data raise the possibility of regulatory interactions between Cmr and CRP Mt at some sites, they strongly suggest that Cmr has additional determinants of specificity that are not obvious from the binding motif.

Motif positions 4 and 13 determine Cmr versus CRP Mt binding specificity
We extended the motif analysis to include sequences flanking the 16-bp core binding sites, but did not identify any additional highly conserved nucleotide positions ( Figure 2A). Therefore, we focused on the core DNA binding motifs of the two TFs to understand the molecular basis of Cmr versus CRP Mt motif recognition specificity. We tested the ability of Cmr to bind a well-characterized CRP Mt binding site in the intergenic region of serC-Rv0885 (28) that contains all known critical nucleotide positions required for Cmr binding (Supplementary Figure S1a and Figure 2B). Despite the presence of all the conserved nucleotides at positions 2, 3, 14 and 15, Cmr bound this DNA sequence very weakly (Figure 2C). Single point mutations at nucleotide positions 4(G: C) or 13(C: G) in the CRP Mt binding site resulted in robust Cmr binding to these DNA sequences, and complete loss of CRP Mt binding. Double mutation to 4(G: C) and 13(C: G) further improved Cmr binding to the DNA sequence. Conversely, Cmr binding was decreased by altering nucleotides at positions 4 or 13 in the PRv1675c Cmr binding site to make it better match the conserved CRP Mt binding motif ( Figure 2D). Double mutation of this DNA fragment at motif positions 4 and 13 resulted in complete loss of Cmr binding.
DNA sequences flanking a core binding site can affect the ability of TFs to recognize and bind DNA, despite the lack of conserved sequence motifs in our extended MEME analyses (45)(46)(47). To assess the effect of flanking sequences on the observed differences in DNA binding, we tested the ability of Cmr to bind a hybrid Cmr (flank CRP ) DNA fragment, which contains the WT 16-bp core palindromic Cmr binding site from PRv1675c and flanking sequences from the CRP Mt binding Rv0885c-serC DNA region. Similarly, we the tested the ability of CRP Mt to bind a hybrid CRP Mt (flank Cmr ) DNA fragment containing the WT 16-bp palindromic CRP Mt binding site from Rv0885-serC CRP Mt binding region, with flanking sequences from PRv1675c. Both Cmr and CRP Mt bound the DNA sequences containing their respective core binding nucleotides regardless of the substituted flanking sequences ( Figure 2C and D). Together, these results indicate that nucleotide positions 2, 3, 14 and 15 comprise a core DNA binding sequence needed for binding by both TFs, while positions 4 and 13 determine the specificity of TF binding. Flanking sequences do not affect binding specificity when both discriminator positions contain Cmr's preferred nucleotides (4C and 13G).

Determination of Cmr structure
We solved the crystal structure of Cmr to elucidate factors that differentiate Cmr and CRP Mt DNA binding at the atomic level. Single-wavelength anomalous dispersion (SAD) was used to solve an initial structure of Cmr 13-244, which was then used for molecular replacement to solve the structure of full length Cmr 1-244 at 1.85Å resolution (Figure 3A). Data collection and refinement statistics are listed in Table 1. In Cmr 1-244, two molecules (A and B) formed a dimer in the asymmetric unit. The structure of Cmr 13-244 showed a similar dimer with the dimer axis coincident with the crystal axis. In both structures, residues at the Nterminal were disordered. Cmr 13-244 was ordered from position 21 to 244, while Cmr 1-244 was ordered from positions 20 and 19 to 244, in molecules A and B respectively. In Cmr 1-244, two ordered sulfate ions present in the crystallization buffer form lattice contacts (not shown). Only small differences were observed upon superposition of molecules A and B of Cmr 1-244 with each other (all C ␣ RMSD = 0.655Å), and with Cmr 13-244 (all C ␣ RMSD = 0.772Å and 1.067Å with molecules A and B, respectively). These differences may be related to crystal packing. The structure of Cmr 1-244 was used for further detailed analysis, as it was more complete.

A comparison of Cmr with CRP/FNR family transcription factors
A search using Dali (48) identified members of the CRP/FNR transcriptional regulator family such as Corynebacterium glutamicum GlxR (PDB ID 4BYY) and Mtb CRP Mt (PDB ID: 3H3U) as structural homologs of Cmr. GlxR and CRP Mt share relatively low amino acid sequence identity with Cmr (26 and 24%, respectively) (4,49,50), while GlxR and CRP Mt share 79% sequence identity with each other. Sequence alignments were generated in ClustalW and related figures were colored using ESPript 3.0 (51) ( Figure 3B). Cmr only shares ∼15% amino acid level sequence identity with E. coli Fnr protein, another prototype member of the CRP-FNR superfamily. Furthermore, Cmr has an extended N-terminal region that is not conserved ( Figure 3B).
In this study, we used the structure of CRP Mt in complex with cAMP and DNA (PDB ID: 3MZH) for all struc-tural comparisons with Cmr 1-244, and our naming of secondary structure elements in Cmr 1-244 ( Figure 3A and B) follows that of CRP Mt (50). Cmr and CRP Mt can be aligned by superimposing 140 corresponding C ␣ positions to yield a RMSD of 1.92Å ( Figure 3C). Similar to CRP Mt , each subunit of Cmr contains a large N-terminal domain comprised of a ␤-sheet core, ending with helix B and joined by central helix C to the smaller C-terminal DNA-binding domain which contains the characteristic HTH domain with DNAbinding helices E and F ( Figure 3A and C).

Differences between Cmr and CRP Mt structures
Despite similarities in overall fold, there are significant differences between the structures of Cmr and CRP Mt . The Cterminal domains of Cmr and CRP Mt show the most conservation ( Figure 3B) and superimpose with RMSD value of 1.18Å over 67 corresponding C ␣ positions ( Figure 3A). The N-terminal domains of Cmr and CRP Mt are less con- The structure of Cmr 1-244 is shown as a ribbon diagram with secondary structure elements labeled. As residues from the N-terminal end were disordered, subunit A (colored yellow) shows amino acid residues from Q20 to the C-terminal end and subunit B (colored turquoise) shows residues from G19 to the C-terminal end. Helices E, F, E' and F' constitute the two HTH DNA binding motifs of Cmr; (B) The sequences of Mycobacterium tuberculosis Cmr, Corynebacterium glutamicum GlxR and M. tuberculosis CRP Mt are aligned (adapted from ClustalW and colored using ESPript 3.0 (51). Labeled secondary structure elements for Cmr and CRP Mt are shown above and below the sequences, respectively. Conserved residues are shown in red; gray boxes define residues in alignment, where at least three contiguous C␣ positions are within 3.5Å of each other. The green asterisks mark the amino acid residues in CRP Mt that directly contact cAMP and form the binding pocket. The blue asterisks mark residues of CRP Mt involved in DNA binding specificity. The magenta asterisks mark residues of the Arg-loop in Cmr; (C) In stereo, subunits of Cmr 1-244 (yellow) and CRP Mt (light gray) are drawn as ribbon diagrams and are superimposed. The Cmr subunit only shows amino acid residues from Q20 to the C-terminal end. The first helix of Cmr and CRP Mt are colored orange and black, respectively. The carbon backbone of the Cmr Arg-loop (R93-R96) is colored magenta, arginine side chains drawn as sticks are colored magenta and blue for carbons and nitrogens, respectively; (D) A model of a Cmr 1-244 dimer bound to DNA, based on PDB structure: 3MZH, is shown with protein and nucleic acid atoms drawn as ribbons and sticks, respectively. The subunits of Cmr are colored yellow and dark gray, and the arginine loops are colored in the same scheme as in Figure 3C; where F o and F c denote observe and calculated structure factors, respectively. e R free was calculated using 5% of data excluded from refinement. f Calculated using Molprobity (43).
served than the C-terminal domains, and superimpose with RMSD value of 1.42Å over 88 corresponding C ␣ positions. The extended N-terminal region of Cmr contains helix N1, which is not conserved and adopts a very different topology compared to CRP Mt ( Figure 3A and C). It should be noted that 19 amino acid residues at the N-terminus of Cmr were disordered and are not shown in Figure 3A, C, D and F. Residues 1-19 in Cmr 1-244, which are disordered in the structure, are also predicted to be disordered by RONN (52) and DISOPRED (53). Helices N1 and N1 of Cmr 1-244 lie between the core ␤-sheet scaffold of the N-terminal domain and corresponding helices C and C , respectively. The position of helices N1 and N1 implies that the disordered residues at the N-terminus would be located at or near the surface of the protein where DNA binding occurs, although their function is not known. Helices N1 and N1 also form part of the dimer interface between the two subunits wherein self-association mediated by contacts between helices N1, N3, B, C of subunit A with N1 , N3 , B and C of subunit B buries 1388Å 2 of total surface area. In contrast, the first helix of CRP Mt is not involved in dimerization. When Cmr and CRP Mt are superimposed, helix N1 of Cmr occupies a location corresponding with the ␤4-␤5 hairpin of CRP Mt ( Figure 3C). In each subunit of CRP Mt , cAMP is found bound in a pocket formed between helix C and the N-terminal domain ␤4-␤5 hairpin loop (54). A similar pocket is notably absent in Cmr 1-244 ( Figure 3E). Instead this region of Cmr is transversed by helix N1 which forms core interactions with helix C and the ␤4-␤5 hairpin. Additionally, side chains (S82, R89 and T134) that allow for hydrogen bonding and coordination of cAMP in CRP Mt are not conserved in Cmr where instead the corresponding residues (P112, M119 and A164) are hydrophobic. A clearly identifiable ligand-binding pocket is not observed elsewhere in Cmr 1-244. A solvent exposed 'arginine loop' (hereafter referred to as Arg-loop) located between ␤-strands 4 and 5 of Cmr contains four consecutive arginine residues at positions 93-96. The sequence of this loop is not conserved in CRP Mt , wherein the sequence is DGRE (Figure 3B), and the location of this loop is also not conserved due to different relative positions of ␤4 and ␤5 in the two proteins. Another noteworthy detail is that in Cmr 1-244, the sequence between helices D and E is ten residues shorter than in CRP Mt , forming just one strand where there are two in CRP Mt . The functional significance of this difference is not known.

Modeling DNA binding to Cmr 1-244
A model of Cmr bound to DNA ( Figure 3D the phosphates in the DNA backbone coincides with a sulfate ion bound between helix E and the ␤10-␤11 loop in Cmr 1-244 (not shown). Arginine residues are known to interact with the DNA backbone and the minor groove (55), and this model places the Arg-loops of each Cmr subunit in proximity to regions of the DNA flanking the recognition sequence, suggesting that they may be involved with DNA binding. Arg loop-DNA interactions beyond those formed with the HTH region might explain the larger DNase I footprint of Cmr (34-bp) (26) compared to that of CRP Mt (27-bp) (19) (Supplementary Figure S1b). Other HTH TFs have been shown to interact with DNA through regions of the protein in addition to the HTH motif (56). Disordered residues 1-19 may become ordered upon binding to a partner and serve a role in regulation (57) as they are also located in a position that could allow DNA interactions (Figure 3D).

Additional regions of Cmr are involved in DNA binding
Based on their predicted locations proximal to the DNA binding face of Cmr ( Figure 3D), we reasoned that the Nterminal region and the Arg-loop might contribute to DNA binding. We also considered the effects of residues R28, R35 and R39 in helices N1 and N2, as this region shows the most variation from the CRP Mt structure, and is analogous to the cAMP-binding pocket in CRP Mt . Site-specific mutations were made targeting each of these regions to determine their contributions to DNA binding ( Figure 3F).
C-terminal His6x-tagged WT Cmr and mutant proteins-CmrR93-96A (denoted hereafter as Cmr-Arg loop *), CmrR28A, CmrR35AR39A and Cmr N12 were overexpressed and purified from E. coli. The majority of Cmr WT and mutant proteins appeared as ∼30 kDa bands in a western blot assay, consistent with the predicted molecular weight of the monomeric form of the proteins ( Figure 4A). Another band migrating at ∼60 kDa was also observed with the WT and mutant proteins, and likely represents a dimer. Chemical crosslinking with glutaraldehyde increased the proportion of dimeric and higher molecular weight forms of the protein, indicating that all of the mutant proteins retained the ability to homodimerize in solution and that functional differences observed between the proteins due to large scale destabilization of protein structure are unlikely. However, we note that, unlike the N-terminal region and the Arg-loop, residues R28, R35 and R39 are involved in key interactions between structural elements in the protein. Thus, any mutations to these residues may alter DNA binding through more subtle perturbations of structure in a way that still preserves dimerization. Therefore, we have exercised cautious restraint in inferring results from the R28A and R35A39A mutant proteins, and focused primarily on the N-terminal and Arg-loop regions in this study.

Arg-loop is required to bind 'imperfect' motifs
We tested the ability of the recombinant proteins to bind a 40-bp dsDNA fragment containing a Cmr binding site from the promoter region of cmr (PRv1675c; same DNA fragment used in Figure 2D). Along with the critical core nu-cleotides (2G, 3T, 14A and 15C), this binding site also contains nucleotides 4C and 13G that are preferred by Cmr for robust binding ( Figure 2D). Cmr WT and all mutant proteins bound the PRv1675c DNA probe ( Figure 4B). Next, we compared the binding of the recombinant proteins to a 40-bp dsDNA fragment containing the Cmr binding site from the Rv3063 intragenic region. This binding site contains all the critical core nucleotides but has a less preferred 13C base, which we expected to negatively affect Cmr's DNA binding ability ( Figure 2D). Robust binding was observed with Cmr WT, CmrN 12 and CmrR28A proteins, but Cmr R35AR39A displayed very weak binding and the Cmr-Arg loop * protein failed to bind this DNA fragment ( Figure 4B). None of the proteins bound the negative control fragment in which the Cmr binding site was scrambled.
Based on the ability of Cmr-Arg loop * to bind PRv1675c but not the Rv3063 DNA fragment, we reasoned that the Arg-loop might facilitate Cmr binding to DNA sequences with less permissive nucleotides in the positions 4 or 13 of the binding site. We tested this hypothesis by comparing the in vitro DNA binding ability of Cmr WT, CmrN 12 and Cmr-Arg loop * proteins to DNA sequences containing either a WT or mutated Cmr binding site from PRv1675c. All three proteins bound and shifted the WT DNA sequence ( Figures 4B and 5). While Cmr WT and CmrN 12 proteins also retained the ability to bind the singly mutated DNA sequences (4C:G or 13G:C), Cmr-Arg loop * failed to bind the mutated sequences. These results show that the Arg-loop interaction is crucial to Cmr's ability to bind DNA when the core binding site contains less permissive nucleotides at positions 4 or 13. We also tested the ability of Cmr WT and mutant proteins to bind the Cmr (flank CRP ) DNA fragment to control for the contribution of flanking sequences to Cmr-Arg loop *'s inability to bind the mutated sequences. Cmr-Arg loop * bound the Cmr (flank CRP ) DNA sequence, suggesting that the specificity determinants are contained within the core DNA sequence motif rather than the flanking sequences.

DNA bending by Cmr
CRP Ec and CRP Mt bend DNA upon binding (20,58), so we investigated the ability of Cmr to bend DNA using five identically sized fragments in which the Cmr binding site is positioned at different distances relative to the center (schematic in Figure 6A). Typically, protein-DNA complexes with the binding site most centrally located have the slowest electrophoretic mobility, and the complexes with binding sites farthest away from the center migrate fastest through a nondenaturing gel, if DNA bending occurs at the TF binding site. We noted that identically sized DNA fragments from PRv1675c region migrated slightly differently in the native gel, even in the absence of protein, suggesting some natural bending in this region ( Figure 6B).
Surprisingly, Cmr WT caused an atypical DNA migration pattern consistent with an asymmetric bend at the PRv1675c binding site ( Figure 6B). Similar migration anomalies were also observed with Cmr N12 or Cmr-Arg loop * in complex with the DNA fragments from the PRv1675c region. With the exception of DNA fragments D and E, Cmr-Arg loop *-DNA complexes migrated much  faster than corresponding Cmr WT-DNA complexes. This suggests that the Arg-loop's interaction with the DNA exerts specific conformational effects on the DNA-protein complex when the binding site is distal to the left end of the DNA probe. The change in charge due to substitution of four arginine residues with alanine in Cmr-Arg loop * is unlikely to be the reason for the faster electrophoretic mobility of Cmr-Arg loop *-DNA complexes A, B and C, as DNA probes D and E in complex with Cmr WT or Cmr-Arg loop * protein migrate similarly through the gel. Rather, altered rigidity and/or shape due to the additional Arg loop contacts may explain this migration anomaly.
We performed a similar experiment with identically sized DNA fragments containing the Cmr binding site in the Rv3063 region ( Figure 6C). These DNA fragments showed no natural bends in DNA in the absence of Cmr binding ( Figure 6D). In the presence of Cmr WT protein, two distinct bands were observed with DNA probes A and B. However, on addition of Cmr N12, DNA-protein complexes with probes A or B migrated as single bands, similar to what was observed with PRv1675c fragments. As expected, Cmr-Arg loop * did not bind these fragments, as this binding site contains the less preferred nucleotide C at motif position 13 ( Figure 4B). Together, these results demonstrate that Cmr bends DNA upon binding. The interaction of the Arg-loop likely has a conformational effect on the DNA-protein complex that affects its electrophoretic migration. However the Arg-loop is not required for DNA bending, as the atypical migration pattern observed with Cmr WT-DNA complexes is also observed with Cmr-Arg loop *-DNA complexes ( Figure 6B). The role of the N-terminal region is less clear, but differences in migration between Rv3063 DNA-protein complexes containing Cmr N12 versus WT Cmr suggest some impact of the N-terminal region on Cmr's DNA interaction.

CmrN 12 binds DNA with higher affinity than Cmr WT
CmrN 12 showed stronger binding to the Rv3063 and PRv1675c DNA fragments, whereas Cmr-Arg loop * showed weaker binding to the PRv1675c fragment and no binding to Rv3063 DNA fragments, compared to the WT protein ( Figures 4B and 5). We determined the binding affinities (K d ) of Cmr WT, CmrN 12 and Cmr-Arg loop * proteins to the 40-bp dsDNA fragment from PRv1675c (Figure 7). CmrN 12 bound DNA with ∼ 3-fold higher affinity than WT protein, whereas Cmr-Arg loop * bound DNA with ∼2fold lower affinity than WT (K d of Cmr WT = 8.3 ± 0.5 nM; K d of CmrN 12 = 2.7 ± 0.1 nM; K d of Cmr-Arg loop * = 13.3 ± 0.9 nM). We were also interested to note that Cmr WT, CmrN 12 and Cmr-Arg loop * bound the DNA sequence containing a single Cmr binding site with a Hill coefficient (h) ≥ 2, indicating strong positive cooperativity in DNA binding. Such strong positive cooperativity has not been reported for CRP Ec or CRP Mt (19,30), consistent with our finding of Hill coefficients of 1.2 for CRP Ec and 1.3 for CRP Mt (Supplementary Figure S2).

Model for DNA binding by Cmr using HTH domain and Argloop
We propose a model of DNA binding by Cmr where, in addition to the core DNA binding contacts made by the HTH domain, the Arg-loop makes supplementary contacts with the sequences flanking the core-binding site ( Figure 8A). Cmr may bend the DNA upon binding in such a way that the determinant nucleotides are positioned to make contacts with the amino acids in the HTH. This bending would also bring the flanking DNA sequences in close proximity to the Arg-loop. It is possible that the HTH mediated binding is sufficient at a binding site that contains all preferred nucleotides, and the Arg-loop contacts serve an accessory purpose. However, these accessory contacts may become critical when the DNA binding site contains less preferred nucleotides, such as 4G or 13C, which cannot be accommodated well by Cmr's HTH.

DISCUSSION
The structure-guided functional analyses in this study provide important new insights into the biology of a TF that contributes to Mtb virulence and pathogenesis. We found that a DNA binding motif derived from Cmr's genomic binding sites does not fully reflect Cmr's high degree of selectivity for DNA binding interactions. Rather, Cmr's DNA binding specificity is determined by multiple levels of DNA interaction, allowing it to recognize subtle differences in DNA binding sites with remarkable specificity. This unexpected complexity likely masks some of Cmr's positional se-quence preferences when the DNA binding motif is derived cumulatively from multiple types of binding sites throughout the genome.
These studies also identified novel structural features of Cmr involved in DNA binding in addition to the HTH DNA binding domain characteristic of the CRP/FNR family. These features include an unusual N-terminal domain and an arginine rich loop, not found in prototype family members CRP Ec , FNR or CRP Mt , distinguish Cmr as a novel member of the CRP/FNR family. The unique fold of Cmr also indicates a more distant evolutionary relationship with CRP/FNR TFs, as suggested previously by a phylogenetic study (6). This functional complexity may provide Cmr with the flexibility to simultaneously integrate multiple signals and modulate its DNA interactions in response to different inputs, including interactions with other TFs, such as those reported for DosR (26,27).

Additional structural features involved in DNA binding
The position of the Arg-loop proximal to the HTH domain is consistent with direct Arg-loop-DNA interaction ( Figure  3D), as is the observation that the Arg-loop is needed for Cmr binding at sites that contain a less preferred nucleotide at motif position 4 or 13 ( Figures 4B, 5 and 7C). However, it is not known whether the Arg-loop contacts specific nucleotides in the DNA sequences flanking the core-binding site. Analysis of Cmr binding sites from the prior ChIP-seq studies (24,26) failed to identify any additional conserved nucleotides in the sequences that flank the core-binding sites (Supplementary Figure S1a and unpublished studies). Also, replacing the DNA sequences flanking the core-binding site had no significant impact on Cmr's DNA binding specificity in the presence of the preferred discriminator bases ( Figure  5). An alternate possibility is that the Arg-loop recognizes local DNA shape. Arginine residues have been associated with DNA-shape based interactions by other TFs, particularly in the minor groove (55). Our results suggest that this additional Arg-loop-DNA interaction becomes important only when one of the DNA binding helices lacks contact with a preferred discriminator base, so future studies will address questions regarding the nature of this contact in the context of different types of core binding sites.
The role of the Cmr N-terminus in DNA binding is not clear. CmrN 12 bound DNA with higher affinity than WT and Cmr-Arg loop * proteins ( Figure 7B), suggesting that the N-terminal region restricts the DNA binding activity of the full-length protein. Consistent with our structural data, DISOPRED (53) and RONN (52) predict residues 1-19 in the N-terminal region of Cmr to be disordered and potentially involved in protein-protein interaction (data not shown). Intrinsically disordered protein domains are being increasingly identified in eukaryotes, prokaryotes and archaea (59). Recent studies suggest a higher prevalence of disordered proteins in bacteria with high GC genomes (59), such as Mtb. It has been proposed that the structural flexibility associated with disordered regions allows them to adopt different conformations based on their physiological environments, facilitating interactions with diverse protein partners (60,61). We speculate that the N-terminal region of Cmr serves a biochemical regulatory role in vivo by modulating Cmr's binding to specific DNA sites in response to physiological signals. Such modulatory signals could include co-factor binding or post-translational modifications that affect binding affinity and/or specificity.

Balancing specificity and plasticity of DNA binding
Cmr failed to bind some CRP Mt binding sites based on the discriminatory nucleotides at positions 4 and 13 of the MEME motif ( Figure 2C). This inability of Cmr to bind CRP Mt binding sites could in part be attributed to the differences in the HTH DNA binding domain of the two TFs. Three residues (Arg188, Glu189 and Lys193) in the HTH DNA binding domains of CRP Mt make direct DNA contacts with the critical nucleotides in the core binding motif ( Figure 8B). The Glu189 residue on each subunit of homodimeric CRP Mt contacts nucleotide 4G or 13C in the motif DNA sequence (20,21,58). However, Glu189 is replaced by Pro209 in Cmr's HTH domain, which likely results in an unfavorable interaction between the Cmr HTH domain and the DNA probe in which motif positions 4 or 13 are G and C, respectively ( Figure 2).
Both the structure and experimental results indicate that the Arg-loop supports the DNA binding function of the HTH domain in Cmr. The Arg-loop-DNA interaction be-comes particularly critical to the DNA binding ability of Cmr at binding sites that contain less preferred nucleotides at motif position 4 or 13 ( Figures 4B and 5). This additional interaction with DNA via the Arg loop also confers plasticity of DNA binding to Cmr, which is more tolerant than CRP Mt to changes at motif positions 4 and 13. Single point mutations at either position negatively affect Cmr's binding, but do not completely eliminate it ( Figure 2D). In contrast, mutating positions 4 or 13 causes complete loss of CRP Mt binding to the mutated sequence ( Figure 2C). The Arg loop, present only in Cmr and absent from CRP Mt , is required to tolerate changes at these nucleotide positions ( Figure 5). Different mechanisms are used by other TFs to distinguish between closely related sequences, or in some cases broaden the binding site repertoire. For example, the Drosophila genome encodes eight Hox proteins that recognize and bind identical binding sites (62,63). However, interaction of these Hox proteins with in vivo co-factors confers a 'latent specificity' to the proteins, allowing them to differentiate between sites that vary only at two central nucleotide positions (64,65). In E. coli, ligation of a metal cluster by IscR relieves an unfavorable DNA-protein interaction by repositioning a single amino acid residue. This broadens the DNA binding specificity of IscR, allowing it to bind two different types of motifs (66). Together, the HTH and the Arg-loop in Cmr allow for specificity by recognizing single nucleotide changes in the binding site, as well as plasticity, because the additional Arg-loop-DNA interaction allows Cmr to tolerate some changes at the discriminatory positions of the binding site. Further studies will address the extent to which this enhanced core site binding flexibility is counterbalanced by unknown determinants of Arg-loop-DNA interactions that may restrict Cmr's binding profile in response to factors other than the core binding sequence. CRP Ec and Fnr have shown reciprocal binding and activation of transcription at some promoters (67). The overlap observed between CRP Mt and Cmr binding sites (Figure 1) raises the possibility of co-regulation of some genes by both these TFs. Future studies focusing on the interaction of Cmr with the transcriptional machinery, particularly at promoters where Cmr and CRP Mt binding sites are overlapping, may provide further insights into the mechanism of transcriptional regulation by Cmr and potential coregulation of genes by Cmr and CRP Mt .

Cmr bends DNA asymmetrically
We found that Cmr binding to its cognate site induces an asymmetrical bend in the DNA (Figure 6), which contrasts with the symmetrical bending caused by CRP Mt (20) and CRP Ec protein (58). Many prokaryotic as well as eukaryotic DNA binding proteins bend DNA upon binding (68)(69)(70)(71)(72)(73)(74), but the reason for this asymmetrical bending with Cmr binding is not clear. Proximal sequences (∼3-7 bp) immediately surrounding the core-binding site tend to influence TF binding more than the distal sequences (75). Swapping the flanking sequences around the core DNA binding motif reversed the direction of the asymmetry (data not shown), suggesting that the flanking sequences influence Cmr's asymmetric DNA bending pattern. The extent to which G/C content of the DNA immediately adjacent the 16-bp core-binding site contributes to the asymmetric bending of DNA upon Cmr binding is not clear ( Figure  6). A DNA probe that contains flanking sequences ∼50% G/C showed a pattern of gel mobility with a mixture of symmetrical and asymmetrical DNA bending ( Figure 6D), although the symmetrical migration required the entire Nterminal domain. As the nucleotide sequence of a DNA fragment contributes to the DNA topology, it is possible that Cmr recognizes DNA shape motifs in the flanking sequences as an additional method of binding site recognition. Such 'indirect readout' mechanisms that facilitate DNA-proteins interaction have been described previously (76)(77)(78)(79).

Cooperative DNA binding suggests a unique mechanism of DNA binding
Cmr WT and mutant proteins displayed strong cooperativity in binding to DNA (Hill coefficient ≈2) (80), whereas DNA binding experiments using CRP Mt and CRP Ec showed much less cooperativity (Hill coefficient of 1.3 and 1.2, respectively) ( Figure 7 and Supplementary Figure S2). This divergence in cooperativity may reflect mechanistic differences in DNA binding by Cmr, compared to CRP Ec and CRP Mt , which are thought to dimerize prior to DNA binding (54,58,67,81). In the case of E. coli Fnr, only the homodimer is capable of binding DNA, and the monomer-dimer equilibrium is controlled by oxygen availability via the degradation/assembly of an oxygen sensitive [4Fe-4S] cluster (82).
Cooperative binding of homodimeric TFs commonly occurs when binding of the TF to one site positively facilitates the binding of a second dimer to another binding site (83)(84)(85). However, the 40-bp DNA probes used in our experiments (Figure 7 and Supplementary Figure S2) each contain only a single binding site (i.e. two-palindromic half sites). Therefore, we propose a model in which binding of monomeric Cmr to a palindromic half-site on the DNA facilitates the binding of the second subunit. In this case, DNA binding would both facilitate and stabilize Cmr dimerization. This model is consistent with a Hill coefficient of ∼2, although the Hill coefficient cannot be used to determine stoichiometry. Analytical ultracentrifugation experiments of Cmr (unpublished data) showed that the protein was present in a major and a minor population, with the minor population migrating at a position consistent with it being a higher molecular weight complex than the major population. However, absolute molecular weight calculations were inconclusive; neither peak had a predicted molecular weight that was consistent with an integral number of molecules in the complex, making these measurements unreliable. The presence of 10% glycerol in the buffers, required for protein stability, is likely to be responsible for the difficulty in obtaining accurate molecular weight measurements.
A recent report concluded that Cmr is dimeric based on size exclusion chromatography (27). However, migration of proteins in the size exclusion chromatography column is based on the protein's hydrodynamic size and volume and can be affected by shape of the protein, thereby causing similar sized proteins to elute at different times. This same re-port (27) also presents EMSA data that are consistent with Cmr binding to DNA with positive cooperativity. Together with our structural and biochemical data suggesting that Cmr has disordered protein domains, it is clear that more definitive methods such as size exclusion chromatography coupled with light scattering or analytical ultracentrifugation under different solution conditions will be required to clearly detect the oligomeric state of Cmr in solution.
The hypothesis that homodimerization of Cmr is facilitated and stabilized by DNA is also supported by our initial observation that a single nucleotide change in one palindromic half-site is tolerated well by Cmr ( Figure 2D). It is plausible that a Cmr monomer binds to the preferred half site first, and nucleates the dimerization as well as the interaction of the second monomer to the 'imperfect' half-site, with the additional DNA binding support from the Argloop. We are currently investigating this hypothesis, as it sets Cmr apart from its CRP/FNR family counterparts, which are thought to dimerize prior to DNA binding.

Absence of an identifiable cAMP binding pocket
The X-ray crystal structure of Cmr revealed the lack of an obvious cAMP binding pocket in the protein ( Figure  3), which is consistent with biochemical studies that failed to detect direct cAMP-Cmr interaction (26,27). The helix N1 in Cmr at least partially blocks the region of Cmr that is analogous to the cAMP binding site in CRP Mt . Additionally, the amino acid residues that coordinate cAMP in CRP Mt are not conserved in Cmr ( Figure 3B). Thus, Cmr appears to be better related to other CRP-family TFs like Sdrp and TTHB099 from T. thermophilus that also lack an effector molecule-binding pocket, and show no conservation of the nucleotide binding residues (86,87). Nonetheless, cAMP-dependent differences in Cmr-DNA interaction have been observed in vitro and in vivo (26). The observed differences in electromobility of DNA-protein complexes in the presence of cAMP often occurred when multiple Cmr binding sites were present, so it is possible that a cryptic cAMP-binding pocket is formed upon Cmr multimerization, resulting in the differential electrophoretic mobility of DNA-protein complexes (26). Alternatively, a large conformational change in Cmr upon DNA binding could occur that makes a cAMP-binding pocket accessible, or cAMP might gain access to a binding pocket in Cmr monomers before they dimerize on the DNA. The difference in mobility of DNA-Cmr complexes in the presence of cAMP despite the absence of an obvious cAMP-binding pocket is intriguing, and warrants further investigation.

Dithiol-disulfide linkage between cysteine residues is unlikely
A recent study suggested that Cmr might act as a redox sensor via a dithiol-disulfide linkage between the two cysteine residues C36 and C131 in Cmr (27). However, our structural analysis indicates that interaction between C36 and C131 is highly unlikely, as the two residues are positioned too far apart and only C36 is solvent exposed. The side chain of C36 packs against helix C at the dimerization interface between the two Cmr monomers, so covalent modification of this residue could perturb the structure of the dimer and/or reduce affinity for self-association through changes in protein conformation at the dimer interface. In this regard, we note that mutating residues R35 and R39, which immediately surround the C36 residue, also had negative effects on Cmr's DNA binding ability in our study (Figure 4). We speculate that the non-conserved N-terminal extension of Cmr (residues 1-39) is sensitive to cellular signals, and can modulate DNA binding by altering the protein conformation and/or its ability to dimerize. The N-terminal extension would thus provide a novel regulatory response mechanism for Cmr.
In summary, we have identified novel regions in Cmr that are involved in its DNA binding ability and specificity of recognition. The additional DNA binding regions not only confer specificity but also a plasticity to Cmr's DNA binding. Our data point towards a distinct mode of Cmr binding to DNA, which sets it apart from CRP Ec and CRP Mt , making Cmr a very unusual member of CRP/FNR family of TFs. Future studies are directed towards understanding the role of Cmr in Mtb biology with a focus on mechanisms underlying co-regulation of genes proximal to overlapping Cmr and CRP Mt binding sites.

DATA AVAILABILITY
The atomic coordinates and structure factors for Cmr1-244 and Cmr 13-244 have been deposited into the Protein Data Bank under accession codes 5W5A and 5W5B, respectively. ChIPseq data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (88) and are accessible through GEO Series accession number GSE106135 (https://www.ncbi.nih.gov/geo/query/acc. cgi?acc=GSE106135).