DNA Binding Affinity Research Articles

Stability and dynamics of extradenticle modulates its function

Extradenticle (EXD) is a partner protein of the HOX transcription factors and plays an important role in the development of Drosophila. It confers increased affinity and specificity of DNA-binding to the HOX proteins. However, the DNA-binding homeodomain of EXD has a significantly weaker affinity to DNA compared to the HOX homeodomains. Here, we show that a glycine residue (G290) in the middle of the EXD DNA-binding helix primarily results in this weaker binding. Glycine destabilizes helices. To probe its role in the stability and function of the protein, G290 was mutated to alanine. The intrinsic stability of the DNA-binding helix increased in the G290A mutant as observed by NMR studies and molecular dynamics (MD) simulation. Also, NMR dynamics and MD simulation show that dynamic motions present in the wild-type protein are quenched in the mutant. This in turn resulted in increased stability of the entire homeodomain (ΔΔGG→A of −2.6 kcal/mol). Increased protein stability resulted in three-fold better DNA-binding affinity of the mutant as compared to the wild-type protein. Molecular mechanics with generalized Born and surface area solvation (MMGBSA) analysis of our MD simulation on DNA-bound models of both wild-type and mutant proteins shows that the contribution to binding is enhanced for most of the interface residues in the mutant compared to the wild-type. Interestingly, the flexible N-terminal arm makes more stable contact with the DNA minor groove in the mutant. We found that the two interaction sites i.e. the DNA-binding helix and the unstructured N-terminal arm influence each other via the bound DNA. These results provide an interesting conundrum: alanine at position 290 enhances both the stability and the DNA-binding affinity of the protein, however, evolution prefers glycine at this position. We have provided several plausible explanations for this apparent conundrum. The function of the EXD as a HOX co-factor requires its ability to discriminate similar DNA sequences, which is most likely compromised by the quenching of its flexibility and enhancement of its stability and DNA-binding affinity.

Synthesis, DNA binding and anticancer properties of new Cu(II) and Zn(II) complexes of a Schiff base ligand containing a triphenylphosphonium as a lipophilic cation

A new quinoline-Schiff base ligand (LTPP) containing the lipophilic triphenylphosphonium cation and its complexes [Cu(LTPP)Cl]Cl and [Zn(LTPP)Cl]Cl were synthesized and characterized. The structures of the ligand and metal complexes were characterized by FTIR, elemental analysis, NMR (for ligand), UV–Vis absorption and fluorescence spectroscopies. DNA binding interactions of the ligand and its metal complexes were investigated by UV–vis absorption and fluorescence spectroscopies as well as viscosity measurements. The DNA binding affinity (Kb) for the ligand and complexes [Cu(LTPP)Cl]Cl and [Zn(LTPP)Cl]Cl) obtained from absorption spectra were found as 4.33, 4.66 and 6.01 (×105 M−1), respectively. The ligand and its Cu(II) complex showed similar propensities to interact with DNA, yet the DNA binding affinity of the Zn(II) complex was relatively higher than free ligand and its Cu(II) complex. Docking studies were conducted in order to further investigate the DNA binding interactions. The ligand and its complexes were screened for their cytotoxic properties towards malignant mesothelioma (H2452) and healthy human umbilical vein endothelial (HUVEC) cells. The Cu(II) and Zn(II) complexes that were loaded into the cells (H2452) confirm their intracellular uptake by fluorescence-based cell imaging. Complex [Zn(LTPP)Cl]Cl emitted fluorescent light even after entering the cell suggesting that the compound can be used for cell tracking purposes. In order to find out whether the compounds we synthesized had the potency to enter human cells, we measured the florescent light emission of H2452 cells treated with the products at their IC50 concentration for 24 h. In vitro antioxidant properties of the ligand and its metal complexes were also studied.

PI3Kδ Inhibition Potentiates Glucocorticoids in B-lymphoblastic Leukemia by Decreasing Receptor Phosphorylation and Enhancing Gene Regulation.

Glucocorticoids are the cornerstone of B-lymphoblastic leukemia (B-ALL) therapy. Because response to glucocorticoids alone predicts overall outcomes for B-ALL, enhancing glucocorticoid potency should improve treatment. We previously showed that inhibition of the lymphoid-restricted PI3Kδ with idelalisib enhances glucocorticoid activity in B-ALL cells. Here, we show that idelalisib enhances glucocorticoid potency in 90% of primary B-ALL specimens and is most pronounced at sub-saturating doses of glucocorticoids near the EC50. Potentiation is associated with enhanced regulation of all glucocorticoid-regulated genes, including genes that drive B-ALL cell death. Idelalisib reduces phosphorylation of the glucocorticoid receptor (GR) at PI3Kδ/MAPK1 (ERK2) targets S203 and S226. Ablation of these phospho-acceptor sites enhances sensitivity to glucocorticoids with ablation of S226 in particular reducing synergy. We also show that phosphorylation of S226 reduces the affinity of GR for DNA in vitro. We propose that PI3Kδ inhibition improves glucocorticoid efficacy in B-ALL in part by decreasing GR phosphorylation, increasing DNA binding affinity, and enhancing downstream gene regulation. This mechanism and the response of patient specimens suggest that idelalisib will benefit most patients with B-ALL, but particularly patients with less responsive, including high-risk, disease. This combination is also promising for the development of less toxic glucocorticoid-sparing therapies.

Impact of polβ/XRCC1 Interaction Variants on the Efficiency of Nick Sealing by DNA Ligase IIIα in the Base Excision Repair Pathway

Base excision repair (BER) requires a coordination from gap filling by DNA polymerase (pol) β to subsequent nick sealing by DNA ligase (LIG) IIIα at downstream steps of the repair pathway. X-ray cross-complementing protein 1 (XRCC1), a non-enzymatic scaffolding protein, forms repair complexes with polβ and LIGIIIα. Yet, the impact of the polβ mutations that affect XRCC1 interaction and protein stability on the repair pathway coordination during nick sealing by LIGIIIα remains unknown. Our results show that the polβ colon cancer-associated variant T304 exhibits a reduced interaction with XRCC1 and the mutations in the interaction interface of V303 loop (L301R/V303R/V306R) and at the lysine residues (K206A/K244A) that prevent ubiquitin-mediated degradation of the protein exhibit a diminished repair protein complex formation with XRCC1. Furthermore, we demonstrate no significant effect on gap and nick DNA binding affinity of wild-type polβ by these mutations. Finally, our results reveal that XRCC1 leads to an efficient channeling of nick repair products after nucleotide incorporation by polβ variants to LIGIIIα, which is compromised by the L301R/V303R/V306R and K206A/K244A mutations. Overall, our findings provide insight into how the mutations in the polβ/XRCC1 interface and the regions affecting protein stability could dictate accurate BER pathway coordination at the downstream steps involving nick sealing by LIGIIIα.

Structural engineering of antimicrobials for optimal broad-spectrum activity

Antimicrobial materials are a crucial component in eradicating and managing the spread of infectious diseases. They are expected to act on a broad-spectrum of microbes, including emerging pathogens which could cause the next Disease X. Herein, we reassessed a series of antimicrobial imidazolium polymers on our shelves and uncovered extended functionality through dual modes of action. By redesigning their structures, a truly broad-spectrum antimicrobial material with optimized activity against bacteria (G ​+ve, G -ve) and fungi, as well as enveloped and non-enveloped viruses was developed. We demonstrated that the imidazolium polymer exhibits dual modes of function against microbes: targeting the microbial membrane and binding DNA. The latter DNA binding affinity was found to be key against non-enveloped viruses. With this insight, we designed small molecule compounds that exhibited optimum broad-spectrum antimicrobial activity and excellent efficacy against ESKAPE group of pathogens that are responsible for some of the deadliest nosocomial infections worldwide. Our results could also shed light on the design of broad-spectrum antimicrobial compounds against Disease X.

Lysine acetylation regulates the AT-rich DNA possession ability of H-NS.

H-NS, the histone-like nucleoid-structuring protein in bacteria, regulates the stability of the bacterial genome by inhibiting the transcription of horizontally transferred genes, such as the type III and type VI secretion systems (T3/T6SS). While eukaryotic histone posttranslational modifications (PTMs) have been extensively studied, little is known about prokaryotic H-NS PTMs. Here, we report that the acetylation of H-NS attenuates its ability to silence horizontally transferred genes in response to amino acid nutrition and immune metabolites. Moreover, LC-MS/MS profiling showed that the acetyllysine sites of H-NS and K120 are indispensable for its DNA-binding ability. Acetylation of K120 leads to a low binding affinity for DNA and enhances T3/T6SS expression. Furthermore, acetylation of K120 impairs the AT-rich DNA recognition ability of H-NS. In addition, lysine acetylation in H-NS modulates in vivo bacterial virulence. These findings reveal the mechanism underlying H-NS PTMs and propose a novel mechanism by which bacteria counteract the xenogeneic silencing of H-NS.

Synthesis and Characterisation of Platinum(II) Diaminocyclohexane Complexes with Pyridine Derivatives as Anticancer Agents.

Cisplatin-type covalent chemotherapeutics are a cornerstone of modern medicinal oncology. However, these drugs remain encumbered with dose-limiting side effects and are susceptible to innate and acquired resistance. The bulk of platinum anticancer research has focused on Cisplatin and its derivatives. Here, we take inspiration from the design of platinum complexes and ligands used successfully with other metals to create six novel complexes. Herein, the synthesis, characterization, DNA binding affinities, and lipophilicity of a series of non-traditional organometallic Pt(II)-complexes are described. These complexes have a basic [Pt(PL)(AL)]Cl2 molecular formula which incorporates either 2-pyrrolidin-2-ylpyridine, 2-(1H-Imidazol-2-yl)pyridine, or 2-(2-pyridyl)benzimidazole as the PL; the AL is resolved diaminocyclohexane. Precursor [Pt(PL)(Cl)2] complexes were also characterized for comparison. While the cytotoxicity and DNA binding properties of the three precursors were unexceptional, the corresponding [Pt(PL)(AL)]2+ complexes were promising; they exhibited different DNA binding interactions compared with Cisplatin but with similar, if not slightly better, cytotoxicity results. Complexes with 2-pyrrolidin-2-ylpyridine or 2-(2-pyridyl)benzimidazole ligands had similar DNA binding properties to those with 2-(1H-Imidazol-2-yl)pyridine ligands but were not as cytotoxic to all cell lines. The variation in activity between cell lines was remarkable and resulted in significant selectivity indices in MCF10A and MCF-7 breast cancer cell lines, compared with previously described similar Pt(II) complexes such as 56MESS.

GATA-3 Dependent Gene Transcription Is Impaired upon HDAC Inhibition

Background: GATA-3 is a lymphoid-lineage specific zinc-finger transcription factor that plays a pivotal role in the development and homeostatic survival of thymic T-cell progenitors and subsets of their mature progeny. Diverse T-cell lymphomas (TCL), derived from conventional (non-malignant) T cells expressing GATA-3, highly express GATA-3 and a GATA-3 dependent transcriptional program, and are generally associated with poor outcomes. Upon binding gene enhancers and promoters, GATA-3 regulates gene transcription, including those with cell-autonomous (e.g. oncogenes, including c-Myc) and non-cell-autonomous (e.g. type 2 cytokines) functions in TCL. Therefore, GATA-3, and the transcriptional program it instigates, is a bona-fide proto-oncogene in these lymphomas. GATA-3 acetylation is dynamically regulated, and its acetylation controls its DNA binding affinity and target gene expression. Therefore, we sought to examine the extent to which HDAC inhibition may alter GATA-3 acetylation and functionally regulate its DNA binding capacity and target gene expression in CTCL. Methods: Integrated GATA-3 ChIP-seq and RNA-seq was performed in CTCL cell lines and primary specimens treated with clinically available HDACi. Complementary strategies were utilized to examine GATA-3/HDAC binding and GATA-3 acetylation in these cells. Results: In order to investigate early transcriptional changes upon HDACi treatment, a well characterized CTCL cell line (H9) and primary Sezary Syndrome (SS) specimens (n=3) were transcriptionally profiled. Gene set enrichment analysis (GSEA) was performed using the differentially expressed genes we identified, and a convergence on relevant T-cell growth and survival pathways was observed. GATA-3 target genes were differentially expressed upon HDACi treatment, and binding sites for GATA-3, and transcription factors (TF) it transcriptionally regulates, were enriched among HDACi responsive genes. Among the 5,510-6,811 transcripts that were differentially expressed upon either belinostat or romidepsin treatment, respectively, ≈6-7% of transcripts were GATA-3 targets. GATA-3 dependent transcripts were significantly altered upon treatment with belinostat or romidepsin in primary SS specimens. We next examined the association between GATA-3 and class I HDACs in CTCL cell lines. HDAC1/HDAC2 and GATA-3 binding was observed, suggesting that HDAC inhibition may lead to GATA-3 hyperacetylation. Treatment with the three clinically available HDACi led to a significant increase in GATA-3 acetylation. As GATA-3 acetylation regulates its ability to bind DNA and regulate transcription, we examined GATA-3 DNA binding using orthogonal approaches, including GATA-3 ChIP-seq, and a significant reduction in GATA-3 binding peaks was observed, culminating in significant changes in the GATA-3 dependent transcriptome. Conclusions: Collectively, these findings demonstrate that GATA-3 hyperacetylation upon HDAC inhibition significantly impairs GATA-3 DNA binding and GATA-3 dependent gene transcription. Impact: GATA-3 and its target genes identify clinically and transcriptionally distinct T-cell lymphomas, including CTCL, that are resistant to conventional chemotherapeutic agents and generally associated with dismal outcomes. Therefore, GATA-3 is a rational, albeit challenging, therapeutic target in these T-cell lymphomas. We have previously explored “upstream” strategies to target GATA-3 by exploiting transcription factors that regulate its expression, and have more recently explored “downstream” strategies by targeting its target genes, including ITK. To the best of our knowledge, the work described here is the first - and a clinically approved - strategy to therapeutically target GATA-3 directly, as HDACi significantly increase GATA-3 acetylation, and by doing so, impair its ability to bind DNA and regulate transcription of its gene targets. Consequently, these findings have significant implications for the interpretation of existing clinical trial datasets and for the development of future combinatorial strategies utilizing an HDACi-containing backbone in GATA-3 driven T-cell lymphomas.

Co-Analysis of Genome-Wide DNA Methylation and Gene Expression Suggests Novel Regulatory Mechanism for RUNX1 in Inv(16) Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is characterized by recurring chromosomal abnormalities that encode oncogenic fusion proteins. Approximately 10% of AML cases are associated with a chromosome 16 inversion [inv(16)(p13q22)] or translocation t(16;16)(p13q22). These chromosomal rearrangements results in the formation of the fusion oncogene CBFB-MYH11 and ultimately the fusion protein CBFβ-SMMHC. Although CBFβ-SMMHC was initially considered a dominant negative repressor of RUNX1, we have recently shown that it works together with RUNX1 to activate gene expression through direct target gene binding. RUNX1 is known to regulate gene expression at both transcriptional and epigenetic levels. Aberrant DNA methylation patterns have been reported across different cancer types including AML. Recent studies have shown that DNA methylation negatively influences RUNX1 DNA binding. We hypothesize that changes to DNA methylation and consequently dysregulated RUNX1 DNA binding contribute to the pathogenesis of inv(16) AML. In this study, we examined the epigenetic and transcriptomic landscapes of CBFB-MYH11 knockin mice and assessed the contribution of the resulting fusion protein CBFβ-SMMHC to RUNX1 DNA target binding. To assess the effects of DNA methylation on RUNX1 DNA binding in vitro, we initially performed fluorescence polarization and electrophoretic mobility shift assays using unmethylated and methylated RUNX1 DNA target sequences. RUNX1, RUNX1/CBFβ and RUNX1/CBFβ-SMMHC showed significantly lower binding affinity for methylated DNA compared to unmethylated DNA probes. To determine genome-wide DNA methylation changes, we performed enzymatic methyl sequencing on sorted Lin -Sca1 -c-Kit + bone marrow cells from control and pre-leukemic inv(16) mice. Inv(16) mice showed a significant genome-wide DNA hypermethylation compared to controls (13,478 hyper- vs 705 hypomethylated regions; q-value < 0.05), with 14% of the differentially methylated regions (DMRs) lying in promoters. Genes associated with these DMRs were enriched for gene ontology (GO) processes related to hematopoiesis, such as myeloid cell homeostasis and erythrocyte differentiation, and DNA replication, such as mitotic DNA replication initiation, among others. In addition, several RUNX1 target genes showed promoter hypermethylation, such as Klf1 or the thrombopoietin receptor Mpl. To understand the impact of aberrant DNA methylation on gene expression, we performed RNA-seq using the same cell population. Gene expression changes were observed in inv(16) mice compared to controls, with 741 genes being upregulated and 278 downregulated from a total of 1019 differentially expressed genes (DEG; absolute fold-change ≥ 2, q-value < 0.05). Enriched GO processes identified from the DEGs were associated with immune responses, such as response to interferon-alpha (and beta) and leukocyte activation involved in inflammatory response. We then intersected the DMR (focusing on promoters) and DEG lists to identify genes whose expression might be directly influenced by DNA methylation changes in inv(16) leukemia. Hypermethylation of promoters was associated with 102 genes, with 67% of them showing downregulation of gene expression in inv(16) mice, whereas hypomethylation was associated with 18 genes with 94% showing upregulation of gene expression. These results show that in inv(16) mice, promoter hypermethylation is associated with downregulation of gene expression, and hypomethylation with increased expression. We also confirmed that several RUNX1 target genes showing promoter hypermethylation also had reduced gene expression in inv(16) mice. The initial analysis suggests that CBFβ-SMMHC expression significantly affects the methylation landscape in mice, with important RUNX1 target genes exhibiting promoter hypermethylation and subsequent downregulation of gene expression. We are currently performing chromatin immunocleavage sequencing using the same cell population to determine RUNX1 binding in vivo. Overall, this study explores a novel regulatory mechanism of RUNX1 function through DNA methylation, and ultimately a new role for RUNX1 in the pathogenesis of inv(16) AML.

Stochastic nucleosome disassembly mediated by remodelers and histone fragmentation.

We construct and analyze monomeric and multimeric models of the stochastic disassembly of a single nucleosome. Our monomeric model predicts the time needed for a number of histone-DNA contacts to spontaneously break, leading to dissociation of a non-fragmented histone from DNA. The dissociation process can be facilitated by DNA binding proteins or processing molecular motors that compete with histones for histone-DNA contact sites. Eigenvalue analysis of the corresponding master equation allows us to evaluate histone detachment times under both spontaneous detachment and protein-facilitated processes. We find that competitive DNA binding of remodeling proteins can significantly reduce the typical detachment time but only if these remodelers have DNA-binding affinities comparable to those of histone-DNA contact sites. In the presence of processive motors, the histone detachment rate is shown to be proportional to the product of the histone single-bond dissociation constant and the speed of motor protein procession. Our simple intact-histone model is then extended to allow for multimeric nucleosome kinetics that reveal additional pathways of disassembly. In addition to a dependence of complete disassembly times on subunit-DNA contact energies, we show how histone subunit concentrations in bulk solutions can mediate the disassembly process by rescuing partially disassembled nucleosomes. Moreover, our kinetic model predicts that remodeler binding can also bias certain pathways of nucleosome disassembly, with higher remodeler binding rates favoring intact-histone detachment.

Activity of the pleiotropic drug resistance transcription factors Pdr1p and Pdr3p is modulated by binding site flanking sequences.

The transcription factors Pdr1p and Pdr3p regulate pleiotropic drug resistance (PDR) in Saccharomyces cerevisiae via the PDR responsive elements (PDREs) to modulate gene expression. However, the exact mechanisms underlying the differences in their regulons remain unclear. Employing genomic occupancy profiling (CUT&RUN), binding assays, and transcription studies, we characterized the differences in sequence specificity between transcription factors. Findings reveal distinct preferences for core PDRE sequences and the flanking sequences for both proteins. While flanking sequences moderately alter DNA binding affinity, they significantly impact Pdr1/3p transcriptional activity. Notably, both proteins demonstrated the ability to bind half sites, showing potential enhancement of transcription from adjacent PDREs. This insight sheds light on ways Pdr1/3p can differentially regulate PDR.

Apoptosis-inducing, anti-angiogenic and anti-migratory effects of a dinuclear Pd(II) complex on breast cancer: A promising novel compound

Because of the high mortality and morbidity rate of breast cancer, successful management of the disease requires synthesis of novel compounds. To this end, ongoing attempts to create new candidates include synthesis of multinuclear metal complexes. The high DNA binding affinity and cytotoxic activity of these complexes makes them promising as breast cancer treatments. This study investigated anti-growth/cytotoxic effect of the dinuclear Pd(II) complex on breast cancer cell lines (MCF-7, MDA-MB-231) using various methods of staining, flow cytometry, and immunoblotting. The study conducted colony formation, invasion, and migration assays were to assess the effect of the complex on metastasis. Increased caspase-3/7 levels and positive annexin V staining were observed in both cell lines, proving apoptosis. Altered TNFR1 and TRADD expression with caspase-8 cleavage followed by BCL-2 inactivation with loss of mitochondrial membrane potential confirmed the presence of apoptosis in MCF-7 and MDA-MB-231, regardless of p53 expression status. The results implied anti-migration properties. Finally, the study used the CAM assay to assess antiangiogenic properties and showed that the complex inhibited angiogenesis. The study concluded the dinuclear Pd(II) complex warrants further in vivo experiments to show its potential in the treatment of breast cancer.

New Organotin (IV) Compounds Derived from Dehydroacetic Acid and Thiosemicarbazides: Synthesis, Rational Design, Cytotoxic Evaluation, and Molecular Docking Simulation.

Organotin complexes were prepared through a one-pot reaction with three components by reacting thiosemicarbazide or 4-methyl-3-thiosemicarbazide or 4-phenylthiosemicarbazide, dehydroacetic acid (DHA) and dibutyl, diphenyl, dicyclohexyl, and bis[(trimethylsilyl)methyl]tin(IV) oxides; all complexes were characterized by infrared (IR), ultraviolet-visible (UV-vis), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. The 119Sn NMR revealed chemical shifts corresponding to a pentacoordinated environment in solution. The X-ray crystallography of the two complexes evidenced the formation of monomeric complexes with a pentacoordinated geometry around tin via three donor atoms from the ligand, the sulfur of the thiol, the nitrogen of the imine group, and the oxygen of the pyran ring. The geometries of the five-coordinated complexes 3a (Bu2SnL3), 3c (Ph2SnL3), and 3d (Cy2SnL3) acid were intermediate between square pyramidal and trigonal bipyramidal, and complex 1a (Bu2SnL1) adopted a bipyramidal trigonal geometry (BPT). The sulforhodamine B assay assessed the cytotoxicity of organotin(IV) complexes against the MDA-MB-231 and MCF-7 (human mammary adenocarcinoma) cell lines and one normal COS-7 (African green monkey kidney fibroblast). The IC50 values evidenced a significant antiproliferative effect on cancer cells; the complexes were more potent than the positive cisplatin control and the corresponding ligands, dehydroacetic acid thiosemicarbazone (L1), dehydroacetic acid-N(4)-methylthiosemicarbazone (L2), and dehydroacetic acid-N(4)-phenylthiosemicarbazone (L3). The IC50 values also indicated that the organotin(IV) complexes were more cytotoxic against the triple-negative breast cell line MDA-MB-231 than MCF-7, inducing significant morphological alterations. The interactions of organotin(IV) 1c (Ph2SnL1), 1d (Cy2SnL1), and 1e (((CH3)3SiCH2)2SnL1) were evaluated with ss-DNA by fluorescence; intensity changes of the fluorescence were indicative of the displacement of ethidium bromide (EB), confirming the interaction of the organotin(IV) complexes with ss-DNA; the results showed a DNA binding affinity. The thermodynamic parameters obtained through isothermal titration calorimetry showed that the interaction of 1c (Ph2SnL1), with ss-ADN, was exothermic. Molecular docking studies also demonstrated that the organotin(IV) complexes were intercalated in DNA by conventional hydrogen bonds, carbon-hydrogen bonds, and π-alkyl interactions. These complexes furthermore showed a greater affinity towards DNA than cisplatin.

TetR and OmpR family regulators in natural product biosynthesis and resistance.

This article provides a comprehensive review and sequence-structure analysis of transcription regulator (TR) families, TetR and OmpR/PhoB, involved in specialized secondary metabolite (SSM) biosynthesis and resistance. Transcription regulation is a fundamental process, playing a crucial role in orchestrating gene expression to confer a survival advantage in response to frequent environmental stress conditions. This process, coupled with signal sensing, enables bacteria to respond to a diverse range of intra and extracellular signals. Thus, major bacterial signaling systems use a receptor domain to sense chemical stimuli along with an output domain responsible for transcription regulation through DNA-binding. Sensory and output domains on a single polypeptide chain (one component system, OCS) allow response to stimuli by allostery, that is, DNA-binding affinity modulation upon signal presence/absence. On the other hand, two component systems (TCSs) allow cross-talk between the sensory and output domains as they are disjoint and transmit information by phosphorelay to mount a response. In both cases, however, TRs play a central role. Biosynthesis of SSMs, which includes antibiotics, is heavily regulated by TRs as it diverts the cell's resources towards the production of these expendable compounds, which also have clinical applications. These TRs have evolved to relay information across specific signals and target genes, thus providing a rich source of unique mechanisms to explore towards addressing the rapid escalation in antimicrobial resistance (AMR). Here, we focus on the TetR and OmpR family TRs, which belong to OCS and TCS, respectively. These TR families are well-known examples of regulators in secondary metabolism and are ubiquitous across different bacteria, as they also participate in a myriad of cellular processes apart from SSM biosynthesis and resistance. As a result, these families exhibit higher sequence divergence, which is also evident from our bioinformatic analysis of 158 389 and 77 437 sequences from TetR and OmpR family TRs, respectively. The analysis of both sequence and structure allowed us to identify novel motifs in addition to the known motifs responsible for TR function and its structural integrity. Understanding the diverse mechanisms employed by these TRs is essential for unraveling the biosynthesis of SSMs. This can also help exploit their regulatory role in biosynthesis for significant pharmaceutical, agricultural, and industrial applications.

Structural and dynamical changes of the Streptococcus gordonii metalloregulatory ScaR protein induced by Mn2+ ion binding

Divalent metal ions are essential micronutrients for many intercellular reactions. Maintaining their homeostasis is necessary for the survival of bacteria. In Streptococcus gordonii, one of the primary colonizers of the tooth surface, the cellular concentration of manganese ions (Mn2+) is regulated by the manganese-sensing transcriptional factor ScaR which controls the expression of proteins involved in manganese homeostasis. To resolve the molecular mechanism through which the binding of Mn2+ ions increases the binding affinity of ScaR to DNA, a variety of computational (QM and MD) and experimental (ITC, DSC, EMSA, EPR, and CD) methods were applied. The computational results showed that Mn2+ binding induces a conformational change in ScaR that primarily affects the position of the DNA binding domains and, consequently, the DNA binding affinity of the protein. In addition, experimental results revealed a 1:4 binding stoichiometry between ScaR dimer and Mn2+ ions, while the computational results showed that the binding of Mn2+ ions in the primary binding sites is sufficient to induce the observed conformational change of ScaR.

Facile fabrication of a Cu(II) 1D zig–zag coordination polymer demonstrating affinity toward DNA molecules

A Cu(II) compound [Cu2(5-nip)2(3-brpy)2·(H2O)2·(DMF)2] (1) was synthesized using 5-nitroisophthalic acid (H25-nip) and 3-bromopyridine (3-brpy) employing slow diffusion technique. Visibly distinct blue-colored crystals of compound 1 were obtained. Single crystal X-ray analysis discloses that compound 1 crystallizes in the triclinic system, having space group P1¯ with cell dimensions a = 12.4491(14) Å, b = 13.6238(15) Å, c = 14.3315(15) Å; α = 68.027(3)°, β = 69.914(3)°, γ = 87.173(3)°and V = 2108.3(4)Å3. The compound contains core metal atom Cu(II) that is hexa-coordinated to 5-nip, 3-brpy, dimethylformamide (DMF), and aqua molecule resulting in one- dimensional coordination polymer (1D CP), propagated via strands of 5-nip bridges. The pyridine-based ligand and aqua ligands are decorated as arms on both sides of Cu(II) centers. The prominence of hydrogen bonding interactions in compound 1 was found which leads to the formation of 1D double-chain strand. The combination of 5-nip and 3-brpy ligands is responsible for the strong affinity towards DNA molecules for compound1. Docking studies have been employed to examine DNA binding affinity. Hirshfeld surface analysis was carried out in order to check out the nature of intermolecular interactions and packing modes of compound 1 in the crystalline state.

Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus.

NifA is a σ54 activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein's central AAA+ domain. In this work, we examine if an additional Cys pair that is part of a C(X)5C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacterium Gluconacetobacter diazotrophicus (Gd) is involved in redox sensing. We hypothesize that the Cys residues' redox state may directly influence the DNA binding domain's DNA binding affinity and/or alter the protein's oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The Kd of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. The Kd of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner.

Mangrove-associated endomycota: diversity and functional significance as a source of novel drug leads.

Endophytic fungi are known for their unprecedented ability to produce novel lead compounds of clinical and pharmaceutical importance. This review focuses on the unexplored fungal diversity associated with mangroves, emphasizing their biodiversity, distribution, and methodological approaches targeting isolation, and identification. Also highlights the bioactive compounds reported from the mangrove fungal endophytes. The compounds are categorized according to their reported biological activities including antimicrobial, antioxidant and cytotoxic property. In addition, protein kinase, α-glucosidase, acetylcholinesterase, tyrosinase inhibition, antiangiogenic, DNA-binding affinity, and calcium/potassium channel blocking activity are also reported. Exploration of these endophytes as a source of pharmacologically important compounds will be highly promising in the wake of emerging antibiotic resistance among pathogens. Thus, the aim of this review is to present a detailed report of mangrove derived endophytic fungi and to open an avenue for researchers to discover the possibilities of exploring these hidden mycota in developing novel drug leads.

Imaging the binding of MECP2 to DNA.

Mutations in the methyl-DNA binding domain of MECP2 cause Rett syndrome; however, distinct mutations are associated with different severity of the disease. Live-cell imaging and single-molecule tracking are sensitive methods to quantify the DNA binding affinity and diffusion dynamics of nuclear proteins. In this issue of Genes & Development, Zhou and colleagues (pp. 883-900) used these imaging methods to quantitatively describe the partial loss of DNA binding resulting from a novel pathological MECP2 mutation with intermediate disease severity. These data demonstrate how single-molecule tracking can advance understanding of the molecular mechanisms connecting MECP2 mutations with Rett syndrome pathophysiology.

Acetylation reprograms MITF target selectivity and residence time

The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome.