Updated understanding of the protein-DNA recognition code used by C2H2 zinc finger proteins.

C2H2 zinc-finger (ZF) proteins form the largest family of DNA-binding transcription factors coded by mammalian genomes. In a typical DNA-binding ZF module, there are twelve residues (numbered from -1 to -12) between the last zinc-coordinating cysteine and the first zinc-coordinating histidine. The established C2H2-ZF "recognition code" suggests that residues at positions -1, -4, and -7 recognize the 5', central, and 3' bases of a DNA base-pair triplet, respectively. Structural studies have highlighted that additional residues at positions -5 and -8 also play roles in specific DNA recognition. The presence of bulky and either charged or polar residues at these five positions determines specificity for given DNA bases: guanine is recognized by arginine, lysine, or histidine; adenine by asparagine or glutamine; thymine or 5-methylcytosine by glutamate; and unmodified cytosine by aspartate. This review discusses recent structural characterizations of C2H2-ZFs that add to our understanding of the principles underlying the C2H2-ZF recognition code.

Quinoline-based compounds can inhibit diverse enzymes that act on DNA.

DNA methylation, as exemplified by cytosine-C5 methylation in mammals and adenine-N6 methylation in bacteria, is a crucial epigenetic mechanism driving numerous vital biological processes. Developing non-nucleoside inhibitors to cause DNA hypomethylation is a high priority, in order to treat a variety of significant medical conditions without the toxicities associated with existing cytidine-based hypomethylating agents. In this study, we have characterized fifteen quinoline-based analogs. Notably, compounds with additions like a methylamine ( 9 ) or methylpiperazine ( 11 ) demonstrate similar low micromolar inhibitory potency against both human DNMT1 (which generates C5-methylcytosine) and Clostridioides difficile CamA (which generates N6-methyladenine). Structurally, compounds 9 and 11 specifically intercalate into CamA-bound DNA via the minor groove, adjacent to the target adenine, leading to a substantial conformational shift that moves the catalytic domain away from the DNA. This study adds to the limited examples of DNA methyltransferases being inhibited by non-nucleotide compounds through DNA intercalation, following the discovery of dicyanopyridine-based inhibitors for DNMT1. Furthermore, our study shows that some of these quinoline-based analogs inhibit other enzymes that act on DNA, such as polymerases and base excision repair glycosylases. Finally, in cancer cells compound 11 elicits DNA damage response via p53 activation. Highlights: Six of fifteen quinoline-based derivatives demonstrated comparable low micromolar inhibitory effects on human cytosine methyltransferase DNMT1, and the bacterial adenine methyltransferases Clostridioides difficile CamA and Caulobacter crescentus CcrM. Compounds 9 and 11 were found to intercalate into a DNA substrate bound by CamA. These quinoline-based derivatives also showed inhibitory activity against various base excision repair DNA glycosylases, and DNA and RNA polymerases. Compound 11 provokes DNA damage response via p53 activation in cancer cells.

C2H2 Zinc Finger Transcription Factors Associated with Hemoglobinopathies.

In humans, specific aberrations in ß-globin results in sickle cell disease and ß-thalassemia, symptoms of which can be ameliorated by increased expression of fetal globin (HbF). Two recent CRISPR-Cas9 screens, centered on ∼1500 annotated sequence-specific DNA binding proteins and performed in a human erythroid cell line that expresses adult hemoglobin, uncovered four groups of candidate regulators of HbF gene expression. They are (1) members of the nucleosome remodeling and deacetylase (NuRD) complex proteins that are already known for HbF control; (2) seven C2H2 zinc finger (ZF) proteins, including some (ZBTB7A and BCL11A) already known for directly silencing the fetal γ-globin genes in adult human erythroid cells; (3) a few other transcription factors of different structural classes that might indirectly influence HbF gene expression; and (4) DNA methyltransferase 1 (DNMT1) that maintains the DNA methylation marks that attract the MBD2-associated NuRD complex to DNA as well as associated histone H3 lysine 9 methylation. Here we briefly discuss the effects of these regulators, particularly C2H2 ZFs, in inducing HbF expression for treating ß-hemoglobin disorders, together with recent advances in developing safe and effective small-molecule therapeutics for the regulation of this well-conserved hemoglobin switch.

A growth-based assay using fluorescent protein emission to screen for S-adenosylmethionine synthetase inhibitors.

The use of cell growth-based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell-based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case l-methionine S-adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing Escherichia coli strain that carries a deletion of metK, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied S-adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species-selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog-selective inhibitors of this enzyme family, some of which target the protozoal human pathogen Cryptosporidium parvum.

Comparative Study of Adenosine Analogs as Inhibitors of Protein Arginine Methyltransferases and a Clostridioides difficile-Specific DNA Adenine Methyltransferase.

S-Adenosyl-l-methionine (SAM) analogs are adaptable tools for studying and therapeutically inhibiting SAM-dependent methyltransferases (MTases). Some MTases play significant roles in host-pathogen interactions, one of which is Clostridioides difficile-specific DNA adenine MTase (CamA). CamA is needed for efficient sporulation and alters persistence in the colon. To discover potent and selective CamA inhibitors, we explored modifications of the solvent-exposed edge of the SAM adenosine moiety. Starting from the two parental compounds (6e and 7), we designed an adenosine analog (11a) carrying a 3-phenylpropyl moiety at the adenine N6-amino group, and a 3-(cyclohexylmethyl guanidine)-ethyl moiety at the sulfur atom off the ribose ring. Compound 11a (IC50 = 0.15 µM) is 10× and 5× more potent against CamA than 6e and 7, respectively. The structure of the CamA-DNA-inhibitor complex revealed that 11a adopts a U-shaped conformation, with the two branches folded toward each other, and the aliphatic and aromatic rings at the two ends interacting with one another. 11a occupies the entire hydrophobic surface (apparently unique to CamA) next to the adenosine binding site. Our work presents a hybrid knowledge-based and fragment-based approach to generating CamA inhibitors that would be chemical agents to examine the mechanism(s) of action and therapeutic potentials of CamA in C. difficile infection.

Structural basis for transcription factor ZBTB7A recognition of DNA and effects of ZBTB7A somatic mutations that occur in human acute myeloid leukemia.

ZBTB7A belongs to a small family of transcription factors having three members in humans (7A, 7B, and 7C). They share a BTB/POZ protein interaction domain at the amino end and a zinc-finger DNA-binding domain at the carboxyl end. They control the transcription of a wide range of genes, having varied functions in hematopoiesis, oncogenesis, and metabolism (in particular glycolysis). ZBTB7A-binding profiles at gene promoters contain a consensus G(a/c)CCC motif, followed by a CCCC sequence in some instances. Structural and mutational investigations suggest that DNA-specific contacts with the four-finger tandem array of ZBTB7A are formed sequentially, initiated from ZF1-ZF2 binding to G(a/c)CCC before spreading to ZF3-ZF4, which bind the DNA backbone and the 3' CCCC sequence, respectively. Here, we studied some mutations found in t(8;21)-positive acute myeloid leukemia patients that occur within the ZBTB7A DNA-binding domain. We determined that these mutations generally impair ZBTB7A DNA binding, with the most severe disruptions resulting from mutations in ZF1 and ZF2, and the least from a frameshift mutation in ZF3 that results in partial mislocalization. Information provided here on ZBTB7A-DNA interactions is likely applicable to ZBTB7B/C, which have overlapping functions with ZBTB7A in controlling primary metabolism.

Systematic Design of Adenosine Analogs as Inhibitors of a Clostridioides difficile-Specific DNA Adenine Methyltransferase Required for Normal Sporulation and Persistence.

Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 µM and KD ∼ 0.2 µM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.

Mediating and maintaining methylation while minimizing mutation: Recent advances on mammalian DNA methyltransferases.

Mammalian genomes are methylated on carbon-5 of many cytosines, mostly in CpG dinucleotides. Methylation patterns are maintained during mitosis via DNMT1, and regulatory factors involved in processes that include histone modifications. Methylation in a sequence longer than CpG can influence the binding of sequence-specific transcription factors, thus affecting gene expression. 5-Methylcytosine deamination results in C-to-T transition. While some mutations are beneficial, most are not; so boosting C-to-T transitions can be dangerous. Given the role of DNMT3A in establishing de novo DNA methylation during development, it is this CpG methylation and deamination that provide the major mutagenic impetus in the DNMT3A gene itself, including the R882H dominant-negative substitution associated with two diseases: germline mutations in DNMT3A overgrowth syndrome, and somatic mutations in clonal hematopoiesis that can initiate acute myeloid leukemia. We discuss recent developments in therapeutics targeting DNMT1, the role of noncatalytic isoform DNMT3B3 in regulating de novo methylation by DNMT3A, and structural characterization of DNMT3A in various configurations.

Differential ETS1 binding to T:G mismatches within a CpG dinucleotide contributes to C-to-T somatic mutation rate of the IDH2 hotspot at codon Arg140.

Cytosine to thymine (C>T) somatic mutation is highly enriched in certain types of cancer, and most commonly occurs via deamination of a 5-methylcytosine (5mC) to thymine, in the context of a CpG dinucleotide. In theory, deamination should occur at equal rates to both 5mC nucleotides on opposite strands. In most cases, the resulting T:G or G:T mismatch can be repaired by thymine DNA glycosylase activities. However, while some hotspot-associated CpG mutations have approximately equal numbers of mutations that resulted either from C>T or G>A in a CpG dinucleotide, many showed strand bias, being skewed toward C>T of the first base pair or G>A of the second base pair. Using the IDH2 Arg140 codon as a case study, we show that the two possible T:G mismatches at the codon-specific CpG site have differing effects on transcription factor ETS1 binding affinity, differentially affecting access of a repair enzyme (MBD4) to the deamination-caused T:G mismatch. Our study thus provides a plausible mechanism for exclusion of repair enzymes by the differential binding of transcription factors affecting the rate at which the antecedent opposite-strand mutations occur.

Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs.

The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2'O-methyladenosine (Am), generating N6, 2'O-dimethyladenosine (m6Am) when Am is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and Am), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2'O-methylated Am over unmodified A nucleosides is due mainly to increased binding affinity for Am. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5'-Am oligonucleotide with approximately the same affinity as that of a cap analog (KM = 0.4 versus 0.3 µM). In addition, PCIF1 methylates the uncapped 5'-Am with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs.

Repurposing epigenetic inhibitors to target the Clostridioides difficile-specific DNA adenine methyltransferase and sporulation regulator CamA.

Epigenetically targeted therapeutic development, particularly for SAM-dependent methylations of DNA, mRNA and histones has been proceeding rapidly for cancer treatments over the past few years. However, this approach has barely begun to be exploited for developing new antibiotics, despite an overwhelming global need to counter antimicrobial resistance. Here, we explore whether SAM analogues, some of which are in (pre)clinical studies as inhibitors of human epigenetic enzymes, can also inhibit Clostridioides difficile-specific DNA adenine methyltransferase (CamA), a sporulation regulator present in all C. difficile genomes sequenced to date, but found in almost no other bacteria. We found that SGC0946 (an inhibitor of DOT1L), JNJ-64619178 (an inhibitor of PRMT5) and SGC8158 (an inhibitor of PRMT7) inhibit CamA enzymatic activity in vitro at low micromolar concentrations. Structural investigation of the ternary complexes of CamA-DNA in the presence of SGC0946 or SGC8158 revealed conformational rearrangements of the N-terminal arm, with no apparent disturbance of the active site. This N-terminal arm and its modulation of exchanges between SAM (the methyl donor) and SAH (the reaction product) during catalysis of methyl transfer are, to date, unique to CamA. Our work presents a substantial first step in generating potent and selective inhibitors of CamA that would serve in the near term as chemical probes to investigate the cellular mechanism(s) of CamA in controlling spore formation and colonization, and eventually as therapeutic antivirulence agents useful in treating C. difficile infection.

Clostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix.

Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile adenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-L-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.

Enzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima.

RNA methylations of varied RNA species (mRNA, tRNA, rRNA, non-coding RNA) generate a range of modified nucleotides, including N6-methyladenosine. Here we study the enzymology of three human RNA methyltransferases that methylate the adenosine amino group in diverse contexts, when it is: the first transcribed nucleotide after the mRNA cap (PCIF1), at position 1832 of 18S rRNA (MettL5-Trm112 complex), and within a hairpin in the 3' UTR of the S-adenosyl-l-methionine synthetase (MettL16). Among these three enzymes, the catalytic efficiency ranges from PCIF1, with the fastest turnover rate of >230 h-1 µM-1 on mRNA cap analog, down to MettL16, which has the lowest rate of ∼3 h-1 µM-1 acting on an RNA hairpin. Both PCIF1 and MettL5 have a binding affinity (Km) of ∼1 µM or less for both substrates of SAM and RNA, whereas MettL16 has significantly lower binding affinities for both (Km >0.4 mM for SAM and ∼10 µM for RNA). The three enzymes are active over a wide pH range (∼5.4-9.4) and have different preferences for ionic strength. Sodium chloride at 200 mM markedly diminished methylation activity of MettL5-Trm112 complex, whereas MettL16 had higher activity in the range of 200 to 500 mM NaCl. Zinc ion inhibited activities of all three enzymes. Together, these results illustrate the diversity of RNA adenosine methyltransferases in their enzymatic mechanisms and substrate specificities and underline the need for assay optimization in their study.

Beta class amino methyltransferases from bacteria to humans: evolution and structural consequences.

S-adenosyl-l-methionine dependent methyltransferases catalyze methyl transfers onto a wide variety of target molecules, including DNA and RNA. We discuss a family of methyltransferases, those that act on the amino groups of adenine or cytosine in DNA, have conserved motifs in a particular order in their amino acid sequence, and are referred to as class beta MTases. Members of this class include M.EcoGII and M.EcoP15I from Escherichia coli, Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM), the MTA1-MTA9 complex from the ciliate Oxytricha, and the mammalian MettL3-MettL14 complex. These methyltransferases all generate N6-methyladenine in DNA, with some members having activity on single-stranded DNA as well as RNA. The beta class of methyltransferases has a unique multimeric feature, forming either homo- or hetero-dimers, allowing the enzyme to use division of labor between two subunits in terms of substrate recognition and methylation. We suggest that M.EcoGII may represent an ancestral form of these enzymes, as its activity is independent of the nucleic acid type (RNA or DNA), its strandedness (single or double), and its sequence (aside from the target adenine).

Structural basis for effects of CpA modifications on C/EBPß binding of DNA.

CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPß binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPß DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPß. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors.

Role for first zinc finger of WT1 in DNA sequence specificity: Denys-Drash syndrome-associated WT1 mutant in ZF1 enhances affinity for a subset of WT1 binding sites.

Wilms tumor protein (WT1) is a Cys2-His2 zinc-finger transcription factor vital for embryonic development of the genitourinary system. The protein contains a C-terminal DNA binding domain with four tandem zinc-fingers (ZF1-4). An alternative splicing of Wt1 can add three additional amino acids-lysine (K), threonine (T) and serine (S)-between ZF3 and ZF4. In the -KTS isoform, ZF2-4 determine the sequence-specificity of DNA binding, whereas the function of ZF1 remains elusive. Three X-ray structures are described here for wild-type -KTS isoform ZF1-4 in complex with its cognate DNA sequence. We observed four unique ZF1 conformations. First, like ZF2-4, ZF1 can be positioned continuously in the DNA major groove forming a 'near-cognate' complex. Second, while ZF2-4 make base-specific interactions with one DNA molecule, ZF1 can interact with a second DNA molecule (or, presumably, two regions of the same DNA molecule). Third, ZF1 can intercalate at the joint of two tail-to-head DNA molecules. If such intercalation occurs on a continuous DNA molecule, it would kink the DNA at the ZF1 binding site. Fourth, two ZF1 units can dimerize. Furthermore, we examined a Denys-Drash syndrome-associated ZF1 mutation (methionine at position 342 is replaced by arginine). This mutation enhances WT1 affinity for a guanine base. X-ray crystallography of the mutant in complex with its preferred sequence revealed the interactions responsible for this affinity change. These results provide insight into the mechanisms of action of WT1, and clarify the fact that ZF1 plays a role in determining sequence specificity of this critical transcription factor.

Complementation of a metK-deficient E. coli strain with heterologous AdoMet synthetase genes.

S-adenosyl-l-methionine (AdoMet) is an essential metabolite, playing a wide variety of metabolic roles. The enzyme that produces AdoMet from l-methionine and ATP (methionine adenosyltransferase, MAT) is thus an attractive target for anti-cancer and antimicrobial agents. It would be very useful to have a system that allows rapid identification of species-specific inhibitors of this essential enzyme. A previously generated E. coli strain, lacking MAT (∆metK) but containing a heterologous AdoMet transporter, was successfully complemented with heterologous metK genes from several bacterial pathogens, as well as with MAT genes from a fungal pathogen and Homo sapiens. The nine tested genes, which vary in both sequence and kinetic properties, all complemented strain MOB1490 well in rich medium. When these strains were grown in glucose minimal medium, growth delays or defects were observed with some specific metK genes, defects that were dramatically reduced if l-methionine was added to the medium.

Methyl-dependent and spatial-specific DNA recognition by the orthologous transcription factors human AP-1 and Epstein-Barr virus Zta.

T: Activator protein 1 (AP-1) is a transcription factor that recognizes two versions of a 7-base pair response element, either 5΄- GAG CA-3΄ or 5΄- GAG CA-3΄ (where M = 5-methylcytosine). These two elements share the feature that 5-methylcytosine and thymine both have a methyl group in the same position, 5-carbon of the pyrimidine, so each of them has two methyl groups at nucleotide positions 1 and 5 from the 5΄ end, resulting in four methyl groups symmetrically positioned in duplex DNA. Epstein-Barr Virus Zta is a key transcriptional regulator of the viral lytic cycle that is homologous to AP-1. Zta recognizes several methylated Zta-response elements, including meZRE1 (5΄- GAG C A-3΄) and meZRE2 (5΄- GAG G A-3΄), where a methylated cytosine occupies one of the inner thymine residues corresponding to the AP-1 element, resulting in the four spatially equivalent methyl groups. Here, we study how AP-1 and Zta recognize these methyl groups within their cognate response elements. These methyl groups are in van der Waals contact with a conserved di-alanine in AP-1 dimer (Ala265 and Ala266 in Jun), or with the corresponding Zta residues Ala185 and Ser186 (via its side chain carbon Cß atom). Furthermore, the two ZRE elements differ at base pair 6 (C:G versus G:C), forming a pseudo-symmetric sequence (meZRE1) or an asymmetric sequence (meZRE2). In vitro DNA binding assays suggest that Zta has high affinity for all four sequences examined, whereas AP-1 has considerably reduced affinity for the asymmetric sequence (meZRE2). We ascribe this difference to Zta Ser186 (a unique residue for Zta) whose side chain hydroxyl oxygen atom interacts with the two half sites differently, whereas the corresponding Ala266 of AP-1 Jun protein lacks such flexibility. Our analyses demonstrate a novel mechanism of 5mC/T recognition in a methylation-dependent, spatial and sequence-specific approach by basic leucine-zipper transcriptional factors.

Evolution of a global regulator: Lrp in four orders of γ-Proteobacteria.

BACKGROUND: Bacterial global regulators each regulate the expression of several hundred genes. In Escherichia coli, the top seven global regulators together control over half of all genes. Leucine-responsive regulatory protein (Lrp) is one of these top seven global regulators. Lrp orthologs are very widely distributed, among both Bacteria and Archaea. Surprisingly, even within the phylum γ-Proteobacteria (which includes E. coli), Lrp is a global regulator in some orders and a local regulator in others. This raises questions about the evolution of Lrp and, more broadly, of global regulators. RESULTS: We examined Lrp sequences from four bacterial orders of the γ-Proteobacteria using phylogenetic and Logo analyses. The orders studied were Enterobacteriales and Vibrionales, in which Lrp plays a global role in tested species; Pasteurellales, in which Lrp is a local regulator in the tested species; and Alteromonadales, an order closely related to the other three but in which Lrp has not yet been studied. For comparison, we analyzed the Lrp paralog AsnC, which in all tested cases is a local regulator. The Lrp and AsnC phylogenetic clusters each divided, as expected, into subclusters representing the Enterobacteriales, Vibrionales, and Pasteuralles. However the Alteromonadales did not yield coherent clusters for either Lrp or AsnC. Logo analysis revealed signatures associated with globally- vs. locally- acting Lrp orthologs, providing testable hypotheses for which portions of Lrp are responsible for a global vs. local role. These candidate regions include both ends of the Lrp polypeptide but not, interestingly, the highly-conserved helix-turn-helix motif responsible for DNA sequence specificity. CONCLUSIONS: Lrp and AsnC have conserved sequence signatures that allow their unambiguous annotation, at least in γ-Proteobacteria. Among Lrp orthologs, specific residues correlated with global vs. local regulatory roles, and can now be tested to determine which are functionally relevant and which simply reflect divergence. In the Alteromonadales, it appears that there are different subgroups of Lrp orthologs, one of which may act globally while the other may act locally. These results suggest experiments to improve our understanding of the evolution of bacterial global regulators.

A surprising range of modified-methionyl S-adenosylmethionine analogues support bacterial growth.

S-Adenosyl-l-methionine (AdoMet) is an essential metabolite, serving in a very wide variety of metabolic reactions. The enzyme that produces AdoMet from l-methionine and ATP (methionine adenosyltransferase, MAT) is thus an attractive target for antimicrobial agents. We previously showed that a variety of methionine analogues are MAT substrates, yielding AdoMet analogues that function in specific methyltransfer reactions. However, this left open the question of whether the modified AdoMet molecules could support bacterial growth, meaning that they functioned in the full range of essential AdoMet-dependent reactions. The answer matters both for insight into the functional flexibility of key metabolic enzymes, and for drug design strategies for both MAT inhibitors and selectively toxic MAT substrates. In this study, methionine analogues were converted in vitro into AdoMet analogues, and tested with an Escherichia coli strain lacking MAT (ΔmetK) but that produces a heterologous AdoMet transporter. Growth that yields viable, morphologically normal cells provides exceptionally robust evidence that the analogue functions in every essential reaction in which AdoMet participates. Overall, the S-adenosylated derivatives of all tested l-methionine analogues modified at the carboxyl moiety, and some others as well, showed in vivo functionality sufficient to allow good growth in both rich and minimal media, with high viability and morphological normality. As the analogues were chosen based on incompatibility with the reactions via which AdoMet is used to produce acylhomoserine lactones (AHLs) for quorum sensing, these results support the possibility of using this route to selectively interfere with AHL biosynthesis without inhibiting bacterial growth.