DNA Binding Energy Research Articles

Evolutionarily conserved and conformationally constrained short peptides might serve as DNA recognition elements in intrinsically disordered regions

Despite recent advances, it is yet not clear how intrinsically disordered regions in proteins recognize their targets without any defined structures. Short linear motifs had been proposed to mediate molecular recognition by disordered regions; however, the underlying structural prerequisite remains elusive. Moreover, the role of short linear motifs in DNA recognition has not been studied. We report a repertoire of short evolutionarily Conserved Recognition Elements (CoREs) in long intrinsically disordered regions, which have very distinct amino-acid propensities from those of known motifs, and exhibit a strong tendency to retain their three-dimensional conformations compared to adjacent regions. The majority of CoREs directly interact with the DNA in the available 3D structures, which is further supported by literature evidence, analyses of ΔΔG values of DNA-binding energies and threading-based prediction of DNA binding potential. CoREs were enriched in cancer-associated missense mutations, further strengthening their functional nature. Significant enrichment of glycines in CoREs and the preference of glycyl ϕ-Ψ values within the left-handed bridge range in the l-disallowed region of the Ramachandran plot suggest that Gly-to-nonGly mutations within CoREs might alter the backbone conformation and consequently the function, a hypothesis that we reconciled using available mutation data. We conclude that CoREs might serve as bait for DNA recognition by long disordered regions and that certain mutations in these peptides can disrupt their DNA binding potential and consequently the protein function. We further hypothesize that the preferred conformations of CoREs and of glycyl residues therein might play an important role in DNA binding. The highly ordered nature of CoREs hints at a therapeutic strategy to inhibit malicious molecular interactions using small molecules mimicking CoRE conformations.

Abstract 18482: Structure Based Prediction of Stability and Affinity Changes in Human NKX2.5 HD Mutants

Background: Cardiac transcription factor, NKX2.5, binds to the target DNA through its homeodomain (HD). To date, nearly 40 mutations in NKX2.5 have been found related to familial congenital heart disease. Homeodomain missense mutations have a high disease penetrance, with pleiotropic cardiac defects compared to missense mutations outside the homeodomain. In in vitro studies, the best correlation with clinical phenotypes and homeodomain mutation resulted from the markedly reduced DNA binding. We recently determined crystal structure of NKX2.5 HD bound to its natural DNA target. Using the structure, we sought to study whether DNA binding and folding energy changes in mutant proteins can be predicted through computational approaches. Methods and results: We used structure based energy prediction to calculate the binding energy and the stability of NKX2.5 mutant proteins. NKX2.5 HD from the high resolution crystal structure (PDB ID: 3RKQ ) was mutated to reflect the 14 genetic and 4 somatic point mutations identified in patients with cardiac anomalies. The HD-DNA models were energy minimized using the program Interface Model. This program utilizes a conservative approach by keeping the backbone structure of the protein and DNA intact while operating only on the sidechain rotamers to achieve the lowest energy state using monte-carlo simulation. Intra-protein and protein-DNA interaction energies of mutant models were calculated and the relative ΔG from the wild type was tabulated. Compared to the wild-type, all the genetic mutants showed reduced DNA binding while the somatic mutations show increased protein stability and DNA binding. The prediction is in good agreement with reported biochemical studies. Conclusion: The high resolution structure enables prediction of energy changes in NKX2.5 pathological mutants compared to wild type. The results well correlate with previously reported experimental data.

Enthalpic switch-points and temperature dependencies of DNA binding and nucleotide incorporation by Pol I DNA polymerases

This study examines the relationship between the DNA binding thermodynamics and the enzymatic activity of the Klenow and Klentaq Pol I DNA polymerases from Escherichia coli and Thermus aquaticus. Both polymerases bind DNA with nanomolar affinity at temperatures down to at least 5°C, but have lower than 1% enzymatic activity at these lower temperatures. For both polymerases it is found that the temperature of onset of significant enzymatic activity corresponds with the temperature where the enthalpy of binding (ΔHbinding) crosses zero (TH) and becomes favorable (negative). This TH/activity upshift temperature is 15°C for Klenow and 30°C for Klentaq. The results indicate that a negative free energy of DNA binding alone is not sufficient to proceed to catalysis, but that the enthalpic versus entropic balance of binding may be a modulator of the temperature dependence of enzymatic function. Analysis of the temperature dependence of the catalytic activity of Klentaq polymerase using expanded Eyring theory yields thermodynamic patterns for ΔG‡, ΔH‡, and TΔS‡ that are highly analogous to those commonly observed for direct DNA binding. Eyring analysis also finds a significant ΔCp‡ of formation of the activated complex, which in turn indicates that the temperature of maximal activity, after which incorporation rate slows with increasing temperature, will correspond with the temperature where the activation enthalpy (ΔH‡) switches from positive to negative.

TherMos: Estimating protein–DNA binding energies from in vivo binding profiles

Accurately characterizing transcription factor (TF)-DNA affinity is a central goal of regulatory genomics. Although thermodynamics provides the most natural language for describing the continuous range of TF-DNA affinity, traditional motif discovery algorithms focus instead on classification paradigms that aim to discriminate ‘bound’ and ‘unbound’ sequences. Moreover, these algorithms do not directly model the distribution of tags in ChIP-seq data. Here, we present a new algorithm named Thermodynamic Modeling of ChIP-seq (TherMos), which directly estimates a position-specific binding energy matrix (PSEM) from ChIP-seq/exo tag profiles. In cross-validation tests on seven genome-wide TF-DNA binding profiles, one of which we generated via ChIP-seq on a complex developing tissue, TherMos predicted quantitative TF-DNA binding with greater accuracy than five well-known algorithms. We experimentally validated TherMos binding energy models for Klf4 and Esrrb, using a novel protocol to measure PSEMs in vitro. Strikingly, our measurements revealed strong non-additivity at multiple positions within the two PSEMs. Among the algorithms tested, only TherMos was able to model the entire binding energy landscape of Klf4 and Esrrb. Our study reveals new insights into the energetics of TF-DNA binding in vivo and provides an accurate first-principles approach to binding energy inference from ChIP-seq and ChIP-exo data.

Theoretical studies on the DNA-intercalator properties of Co(III) polypyridyl complexes

Theoretical studies on the DNA-intercalator properties of Co(III) polypyridyl complexes 1 and 2 have been carried out. First, a sheared whole DNA-complex model to perform the study on DNA-interaction properties of Co(III) polypyridyl complexes was established, by using a combination method of the quantum-chemistry and the molecular mechanics as follows: The full geometric optimizations of these two complexes were carried out in aqueous solution using the restricted B3LYP method and LanL2DZ basis set, then the optimized complexes were docked into DNA-base-pairs using the docking program, and the obtained greater DNA-complex model was sheared to a simplified whole model, and finally the sheared DNA-complex model was exactly computed using the DFT/TDDFT method. Secondly, based on the computations of above-established models, via the analysis of the DNA-binding energies of complexes 1 and 2 as well as the intramolecular hydrogen bonds within the docking models, the trend in DNA-binding affinities, i.e., K b( 2) > K b( 1), can be reasonably explained. Thirdly, the electronic absorption spectra of docking models were also calculated and simulated using the TDDFT method. The simulated absorption spectra are in a satisfying agreement with experimental results, and the intense experimental absorption bands of these Co(Ш) complexes in the presence of DNA are theoretically explained in detail.

Statistical Mechanics of Nucleosomes Constrained by Higher-Order Chromatin Structure.

Eukaryotic DNA is packaged into chromatin: one-dimensional arrays of nucleosomes separated by stretches of linker DNA are folded into 30-nm chromatin fibers which in turn form higher-order structures (Felsenfeld and Groudine in Nature 421:448, 2003). Each nucleosome, the fundamental unit of chromatin, has 147 base pairs (bp) of DNA wrapped around a histone octamer (Richmond and Davey in Nature 423:145, 2003). In order to describe how chromatin fiber formation affects nucleosome positioning and energetics, we have developed a thermodynamic model of finite-size particles with effective nearest-neighbor interactions and arbitrary DNA-binding energies. We show that both one-and two-body interactions can be extracted from one-particle density profiles based on high-throughput maps of in vitro or in vivo nucleosome positions. Although a simpler approach that neglects two-body interactions (even if they are in fact present in the system) can be used to predict sequence determinants of nucleosome positions, the full theory is required to disentangle one- and two-body effects. Finally, we construct a minimal model in which nucleosomes are positioned primarily by steric exclusion and two-body interactions rather than intrinsic histone-DNA sequence preferences. The model reproduces nucleosome occupancy patterns observed over transcribed regions in living cells.

Asymmetric Assembly of Merkel Cell Polyomavirus Large T-Antigen Origin Binding Domains at the Viral Origin

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 Å crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistent with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be ∼ 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein–protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein–protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.

Impact of Covalent Modifications on Binding and Conformational Propensities of Histone Tails

Histone tails are highly flexible N terminal protrusions of histone proteins which help to fold DNA into dense superstructures known as chromatin. On a molecular scale histone tails are polyelectrolites with high degree of conformational disorder, allowing them to function as bio-molecular ”switches”, regulating various genetic regulatory processes via diverse types of covalent modifications. Because of being intrinsically disordered, the structural and dynamical aspects of histone tails are still poorly understood. In this work we have investigated the impact of experimentally well studied covalent modifications on conformational and DNA-binding propensities of H4/H3 histone tails(methylation/acetylation respectively). We carried out long time REMD simulations on wild-type and covalently modified forms of H4/H3 tails in presence of explicit water and ions. Our results demonstrated how covalent perturbations impact the stability of secondary structural elements that are present in the wild-type forms. We reconciled some of our previous results with in vivo experiments by probing DNA-binding energy landscapes of H4 and H3 tails showing that in presence of DNA intrinsic conformational preferences are well preserved, which is manifested in formation of fairly similar secondary structural elements upon binding. Our results also showed that binding free energy gain is sufficiently small(∼3kT) to set dynamic equilibrium between DNA-bound and unbound states. Additionally we have computed the free energy profiles of DNA binding for wild and covalently modified forms finding a correlation between binding strength and change in stability of the chromatin fiber that takes place upon covalent modification of H3/H4 histone tails.

Applying small-scale DNA signatures as an aid in assembling soybean chromosome sequences.

Previous work has established a genomic signature based on relative counts of the 16 possible dinucleotides. Until now, it has been generally accepted that the dinucleotide signature is characteristic of a genome and is relatively homogeneous across a genome. However, we found some local regions of the soybean genome with a signature differing widely from that of the rest of the genome. Those regions were mostly centromeric and pericentromeric, and enriched for repetitive sequences. We found that DNA binding energy also presented large-scale patterns across soybean chromosomes. These two patterns were helpful during assembly and quality control of soybean whole genome shotgun scaffold sequences into chromosome pseudomolecules.

Investigating the Bactericidal Mechanism of Three Novel Histone-Derived Antimicrobial Peptides

Many antimicrobial peptides elicit bacterial death via cell membrane lysis. However, buforin II (BF2), a 21-amino acid peptide derived from histone H2A, is unique due to its hypothesized ability to translocate across cell membranes and interact with bacterial nucleic acids to cause cell death. Since cell-entry peptides, such as BF2, often are effective at lower concentrations than peptides that target membranes, increasing their potential as therapeutics in vivo. To this end, we developed three novel histone-derived antimicrobial peptides based on fragments of histones H2A (Des1), H3 (Des2) and H4 (Des3). These histones were previously found to exhibit translocation behavior. The designed peptides' antimicrobial properties were verified using a radial diffusion assay. In this assay, BF2 exhibited the greatest antimicrobial activity, followed by Des1, Des3 and Des2, respectively. We also measured the absorbance of bacterial cells incubated with these peptides. Based on the similarity between the absorbance versus time trend for the designed peptides with other DNA-binding antimicrobial peptides, such as BF2 and indolicidin, molecular dynamics simulations were used to model the peptides' interactions with nucleic acids. MM-GBSA analyses of the simulations were used to calculate DNA binding energies of individual peptide residues. We used these analyses to create mutant versions of the designed peptides that were predicted to have altered DNA binding. Experimental measurements of the DNA binding and antimicrobial properties of these variants will help us determine whether nucleic acid interactions are important in the bactericidal mechanism of the designed peptides. Ongoing work on the designed peptides is aimed at investigating their translocation behavior in vitro with lipid vesicles and in vivo with bacterial cells using confocal microscopy and fluorescently tagged peptides.

Synthesis and DNA-binding studies of two ruthenium(II) complexes of an intercalating ligand

Two new ruthenium(II) complexes [Ru(bpy) 2(HBT)] 2+ ( 1) and [Ru(phen) 2(HBT)] 2+ ( 2) (bpy = 2,2′-bipyridine; phen = 1,10-phenanthroline; HBT = 11 H, 13 H-4, 5,9,10,12,14-hexaaza-benzo [ b] triphenylene) have been synthesized and characterized by elemental analysis, mass spectra, 1H NMR and cyclic voltammetry. The DNA binding property of the two complexes has been investigated employing absorption spectroscopy, fluorescence spectroscopy and viscosity measurements. Both complexes 1 and 2 have been found to prefer intercalative binding to DNA. The DNA binding constants for the two complexes have been measured to be 5.71 ± 0.20 × 10 7 and 4.65 ± 0.20 × 10 7 M −1 through ethidium bromide displacement method. Molecular modeling studies too indicate that both complexes 1 and 2 prefer intercalative binding to DNA and both these complexes exhibit similar DNA binding energies. Both complexes 1 and 2 bring about photocleavage of plasmid DNA when irradiated at 440 nm.

Describing Delicate Interactions

Quantifying the affinities of interactions in biological networks, particularly transient ones, remains a challenge. Maerkl and Quake describe a high-throughput microfluidic platform that allows the measurement of transient and low-affinity interactions and characterize the DNA binding energy landscapes for four eukaryotic transcription factors. In two cases, the binding specificities were used to predict which genes the transcription factors would bind and likely regulate. S. J. Maerkl, S. R. Quake, A systems approach to measuring the binding energy landscapes of transcription factors. Science 315 , 233-237 (2007). [Abstract] [Full Text]

A Systems Approach to Measuring the Binding Energy Landscapes of Transcription Factors

A major goal of systems biology is to predict the function of biological networks. Although network topologies have been successfully determined in many cases, the quantitative parameters governing these networks generally have not. Measuring affinities of molecular interactions in high-throughput format remains problematic, especially for transient and low-affinity interactions. We describe a high-throughput microfluidic platform that measures such properties on the basis of mechanical trapping of molecular interactions. With this platform we characterized DNA binding energy landscapes for four eukaryotic transcription factors; these landscapes were used to test basic assumptions about transcription factor binding and to predict their in vivo function.

Precise physical models of protein–DNA interaction from high-throughput data

A cell's ability to regulate gene transcription depends in large part on the energy with which transcription factors (TFs) bind their DNA regulatory sites. Obtaining accurate models of this binding energy is therefore an important goal for quantitative biology. In this article, we present a principled likelihood-based approach for inferring physical models of TF-DNA binding energy from the data produced by modern high-throughput binding assays. Central to our analysis is the ability to assess the relative likelihood of different model parameters given experimental observations. We take a unique approach to this problem and show how to compute likelihood without any explicit assumptions about the noise that inevitably corrupts such measurements. Sampling possible choices for model parameters according to this likelihood function, we can then make probabilistic predictions for the identities of binding sites and their physical binding energies. Applying this procedure to previously published data on the Saccharomyces cerevisiae TF Abf1p, we find models of TF binding whose parameters are determined with remarkable precision. Evidence for the accuracy of these models is provided by an astonishing level of phylogenetic conservation in the predicted energies of putative binding sites. Results from in vivo and in vitro experiments also provide highly consistent characterizations of Abf1p, a result that contrasts with a previous analysis of the same data.

Extensive low-affinity transcriptional interactions in the yeast genome

Major experimental and computational efforts are targeted at the characterization of transcriptional networks on a genomic scale. The ultimate goal of many of these studies is to construct networks associating transcription factors with genes via well-defined binding sites. Weaker regulatory interactions other than those occurring at high-affinity binding sites are largely ignored and are not well understood. Here I show that low-affinity interactions are abundant in vivo and quantifiable from current high-throughput ChIP experiments. I develop algorithms that predict DNA-binding energies from sequences and ChIP data across a wide dynamic range of affinities and use them to reveal widespread functionality of low-affinity transcription factor binding. Evolutionary analysis suggests that binding energies of many transcription factors are conserved even in promoters lacking classical binding sites. Gene expression analysis shows that such promoters can generate significant expression. I estimate that while only a small percentage of the genome is strongly regulated by a typical transcription factor, up to an order of magnitude more may be involved in weaker interactions. Low-affinity transcription factor-DNA interaction may therefore be important both evolutionarily and functionally.

Cholesterol and its fatty acid esters in native DNA preparations: lipid analysis, computer simulation of their interaction with DNA and cholesterol binding to immobilized oligodeoxyribonucleotides

Supramolecular DNA complexes were isolated from rat normal cells and murine tumors. The content of DNA-bound lipids (cholesterol and its esters) was determined. The content of cholesterol esters is higher than that of free cholesterol; the lipid content in tumor cells is higher than in normal cells. Using the molecular mechanics approach, it is demonstrated for the first time that cholesterol and its esters with stearic, oleic, linoleic, and linolenic acids bind to the DNA minor groove more strongly than with the major groove. The calculated DNA binding energies of cholesterol and its esters depend on both the number of double bonds in the fatty acid residue and on the DNA nucleotide composition. The formation of stable complexes between cholesterol molecules and d(AT)-rich oligonucleotides was demonstrated using biological microchip containing immobilized octadeoxyribonucleotides.

A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase

Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid-binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid. Now we identify the stage of the ATPase cycle responsible for translocation and unwinding. We show that a rapid directional movement occurs upon helicase binding to DNA in the absence of ATP, resulting in opening of several base pairs. We propose that HCV helicase translocates as a Brownian motor with a simple two-stroke cycle. The directional movement step is fueled by single-stranded DNA binding energy while ATP binding allows for a brief period of random movement that prepares the helicase for the next cycle.

Transcriptional activation mechanisms of the PRM promoter of λ phage

We investigate the transcriptional activity associated with the P RM promoter of λ phage. The probability for formation of a transcriptionally active (open) RNA polymerase–DNA complex is calculated by means of an equilibrium statistical–mechanical model. In particular, we study two different models of the transcriptional activation mechanism when the O R2 site is occupied by a CI dimer (typical for a lysogen) compared to the situation when O R2 is vacant: (1) transcription rate increases (wild-type mechanism) or (2) RNA polymerase becomes stronger (or weaker) bound to DNA (mutant mechanism). By applying experimental determined protein–DNA binding energies we show that these two mechanisms exhibit different characteristics when we study the activity versus CI concentration. We also show that our model may be fitted to in vivo activity data satisfactorily despite that the activated transcription rate is significantly reduced compared to the wild-type value. The model is consistent with experimental determined activities based upon mutations of CI and RNA polymerase.

Kinetics of Protein-DNA Interaction: Facilitated Target Location in Sequence-Dependent Potential

Recognition and binding of specific sites on DNA by proteins is central for many cellular functions such as transcription, replication, and recombination. In the process of recognition, a protein rapidly searches for its specific site on a long DNA molecule and then strongly binds this site. Here we aim to find a mechanism that can provide both a fast search (1–10 s) and high stability of the specific protein-DNA complex ( K d = 10 −15–10 −8 M). Earlier studies have suggested that rapid search involves sliding of the protein along the DNA. Here we consider sliding as a one-dimensional diffusion in a sequence-dependent rough energy landscape. We demonstrate that, despite the landscape’s roughness, rapid search can be achieved if one-dimensional sliding is accompanied by three-dimensional diffusion. We estimate the range of the specific and nonspecific DNA-binding energy required for rapid search and suggest experiments that can test our mechanism. We show that optimal search requires a protein to spend half of its time sliding along the DNA and the other half diffusing in three dimensions. We also establish that, paradoxically, realistic energy functions cannot provide both rapid search and strong binding of a rigid protein. To reconcile these two fundamental requirements we propose a search-and-fold mechanism that involves the coupling of protein binding and partial protein folding. The proposed mechanism has several important biological implications for search in the presence of other proteins and nucleosomes, simultaneous search by several proteins, etc. The proposed mechanism also provides a new framework for interpretation of experimental and structural data on protein-DNA interactions.

Sensitivity of Phage Lambda upon Variations of the Gibbs Free Energy

AbstractWe investigate the sensitivity of production rates (activities) of the regulatory proteins CI (repressor) and Cro at the right operator (OR) of bacteriophage lambda. The DNA binding energies of CI, Cro, and RNA polymerase are perturbed to check the uncertainty of the activity, due to the experimental error, by means of a computational scattering method according to which the binding energies are simultaneously chosen at random around the literature values, with a width corresponding to the experimental error. In a grand canonical ensemble, with the randomly drawn protein‐DNA binding energies as input, we calculate the corresponding activities of the promoters PRM and PR. By repeating this procedure we obtain a mean value of the activity that roughly corresponds to wild‐type (unperturbed) activity. The standard deviation emerging from this scheme, a measure of the sensitivity due to experimental error, is significant (typically >20% relative to wild‐type activity), but still the promoter activities are sufficiently separated to make the switch feasible. We also suggest a new, compact way of presenting repressor and Cro data.