Binding to the Other Side: The AT-Hook DNA-Binding Domain Allows Nuclear Factors to Exploit the DNA Minor Groove
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ABSTRACT:
SUBMITTER: Battista S
PROVIDER: S-EPMC11354804 | biostudies-literature | 2024 Aug
REPOSITORIES: biostudies-literature
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Project description:We report the development of chimeric DNA binding peptides comprising a DNA binding fragment of natural transcription factors (the basic region of a bZIP protein or a monomeric zinc finger module) and an AT-Hook peptide motif. The resulting peptide conjugates display high DNA affinity and excellent sequence selectivity. Furthermore, the AT-Hook motif also favors the cell internalization of the conjugates.
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Project description:The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as 'AT-hooks', are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three 'AT-hook' peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, 'key-locked' conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution.
| S-EPMC8934665 | biostudies-literature
Project description:The energetics for binding of a diphenyl diamidine antitrypanosomal agent CGP 40215A to DNA have been studied by spectroscopy, isothermal titration calorimetry, and surface plasmon resonance biosensor methods. Both amidines are positively charged under experimental conditions, but the linking group for the two phenyl amidines has a pK(a) of 6.3 that is susceptible to a protonation process. Spectroscopic studies indicate an increase of 2.7 pK(a) units in the linking group when the compound binds to an A/T minor-groove site. Calorimetric titrations in different buffers and pH conditions support the proton-linkage process and are in a good agreement with spectroscopic titrations. The two methods established a proton-uptake profile as a function of pH. The exothermic enthalpy of complex formation varies with different pH conditions. The observed binding enthalpy increases as a function of temperature indicating a negative heat capacity change that is typical for DNA minor-groove binders. Solvent accessible surface area calculations suggest that surface burial accounts for about one-half of the observed intrinsic negative heat capacity change. Biosensor and calorimetric experiments indicate that the binding affinities vary with pH values and salt concentrations due to protonation and electrostatic interactions. The surface plasmon resonance binding studies indicate that the charge density per phosphate in DNA hairpins is smaller than that in polymers. Energetic contributions from different factors were also estimated for the ligand/DNA complex.
| S-EPMC1367283 | biostudies-literature
Project description:Heterocyclic diamidines are strong DNA minor-groove binders and have excellent antiparasitic activity. To extend the biological activity of these compounds, a series of arylimidamides (AIAs) analogues, which have better uptake properties in Leishmania and Trypanosoma cruizi than diamidines, was prepared. The binding of the AIAs to DNA was investigated by Tm , fluorescence displacement titration, circular dichroism, DNase I footprinting, biosensor surface plasmon resonance, X-ray crystallography and molecular modeling. These compounds form 1:1 complexes with AT sequences in the DNA minor groove, and the binding strength varies with substituent size, charge and polarity. These substituent-dependent structure and properties provide a SAR that can be used to estimate K values for binding to DNA in this series. The structural results and molecular modeling studies provide an explanation for the differences in binding affinities for AIAs.
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Project description:Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.
| S-EPMC3618862 | biostudies-literature
Project description:High mobility group (HMG) proteins are intrinsically disordered nuclear non-histone chromosomal proteins that play an essential role in many biological processes by regulating the expression of numerous genes in eukaryote cells. HMGA proteins contain three DNA binding motifs, the "AT-hooks", that bind preferentially to AT-rich sequences in the minor groove of B-form DNA. Understanding the interactions of AT-hook domains with DNA is very relevant from a medical point of view because HMGA proteins are involved in different conditions including cancer and parasitic diseases. We present here the first crystal structure (1.40 Å resolution) of the HMGA AT-hook 1 domain, bound to the minor groove of AT-rich DNA. In contrast to AT-hook 3 which bends DNA and shows a larger minor groove widening, AT-hook 1 binds neighbouring DNA molecules and displays moderate widening of DNA upon binding. The binding affinity and thermodynamics of binding were studied in solution with surface plasmon resonance (SPR)-biosensor and isothermal titration calorimetry (ITC) experiments. AT-hook 1 forms an entropy-driven 2:1 complex with (TTAA)2-containing DNA with relatively slow kinetics of association/dissociation. We show that N-phenylbenzamide-derived antikinetoplastid compounds (1-3) bind strongly and specifically to the minor groove of AT-DNA and compete with AT-hook 1 for binding. The central core of the molecule is the basis for the observed sequence selectivity of these compounds. These findings provide clues regarding a possible mode of action of DNA minor groove binding compounds that are relevant to major neglected tropical diseases such as leishmaniasis and trypanosomiasis.
| S-EPMC11526092 | biostudies-literature
Project description:Control of the release properties of drugs has been considered a key factor in the development of drug delivery systems (DDSs). However, drug delivery has limitations including cytotoxicity, low loading efficiency, and burst release. To overcome these challenges, nano or micro-particles have been suggested as carrier systems to deliver chemical drugs. Herein, nano-sized DNA particles (DNAp) were manufactured to deliver netropsin, which is known to bind to DNA minor grooves. The rationally designed particles with exposed rich minor grooves were prepared by DNAp synthesis via rolling circle amplification (RCA). DNAp could load large quantities of netropsin in its minor grooves. An analytical method was also developed for the quantification of netropsin binding to DNAp by UV-visible spectrometry. Moreover, controlled release of netropsin was achieved by forming a layer of Ca2+ on the DNAp (CaDNAp). As a proof of concept, the sustained release of netropsin by CaDNAp highlights the potential of the DNAp-based delivery approach.
| S-EPMC6724015 | biostudies-literature
Project description:Small molecule complexes with DNA that incorporate linking water molecules are rare, and the DB921-DNA complex has provided a unique and well-defined system for analysis of water-mediated binding in the context of a DNA complex. DB921 has a benzimidazole-biphenyl system with terminal amidines that results in a linear conformation that does not possess the appropriate radius of curvature to match the minor groove shape and represents a new paradigm that does not fit the classical model of minor groove interactions. To better understand the role of the bound water molecule observed in the X-ray crystal structure of the DB921 complex, synthetic modifications have been made in the DB921 structure, and the interactions of the new compounds with DNA AT sites have been evaluated with an array of methods, including DNase I footprinting, biosensor-surface plasmon resonance, isothermal titration microcalorimetry, and circular dichroism. The interaction of a key compound, which has the amidine at the phenyl shifted from the para position in DB921 to the meta position, has also been examined by X-ray crystallography. The detailed structural, thermodynamic, and kinetic results provide valuable new information for incorporation of water molecules in the design of new lead scaffolds for targeting DNA in chemical biology and therapeutic applications.
| S-EPMC3165004 | biostudies-literature
Project description:To determine what topological changes antiparasitic heterocyclic dications can have on kinetoplast DNA, we have constructed ligation ladders, with phased A5 and ATATA sequences in the same flanking sequence context, as models. Bending by the A5 tract is observed, as expected, while the ATATA sequence bends DNA very little. Complexes of these DNAs with three diamidines containing either furan, thiophene or selenophene groups flanked by phenylamidines were investigated along with netropsin. With the bent A5 ladder the compounds caused either a slight increase or decrease in the bending angle. Surprisingly, however, with ATATA all of the compounds caused significant bending, to values close to or even greater than the A5 bend angle. Results with a mixed cis sequence, which has one A5 and one ATATA, show that the compounds bend ATATA in the same direction as a reference A5 tract, that is, into the minor groove. These results are interpreted in terms of a groove structure for A5 which is largely pre-organized for a fit to the heterocyclic amidines. With ATATA the groove is intrinsically wider and must close to bind the compounds tightly. The conformational change at the binding site then leads to significant bending of the alternating DNA sequence.
| S-EPMC2760788 | biostudies-literature
Project description:Understanding the molecular basis of ligand-DNA-binding events, and its application to the rational design of novel drugs, requires knowledge of the structural features and forces that drive the corresponding recognition processes. Existing structural evidence on DNA complexation with classical minor groove-directed ligands and the corresponding studies of binding energetics have suggested that this type of binding can be described as a rigid-body association. In contrast, we show here that the binding-coupled conformational changes may be crucial for the interpretation of DNA (hairpin) association with a classical minor groove binder (netropsin). We found that, although the hairpin form is the only accessible state of ligand-free DNA, its association with the ligand may lead to its transition into a duplex conformation. It appears that formation of the fully ligated duplex from the ligand-free hairpin, occurring via two pathways, is enthalpically driven and accompanied by a significant contribution of the hydrophobic effect. Our thermodynamic and structure-based analysis, together with corresponding theoretical studies, shows that none of the predicted binding steps can be considered as a rigid-body association. In this light we anticipate our thermodynamic approach to be the basis of more sophisticated nucleic acid recognition mechanisms, which take into account the dynamic nature of both the nucleic acid and the ligand molecule.
| S-EPMC2241884 | biostudies-literature