Negatively charged, intrinsically disordered regions can accelerate target search by DNA-binding proteins.

In eukaryotes, many DNA/RNA-binding proteins possess intrinsically disordered regions (IDRs) with large negative charge, some of which involve a consecutive sequence of aspartate (D) or glutamate (E) residues. We refer to them as D/E repeats. The functional role of D/E repeats is not well understood, though some of them are known to cause autoinhibition through intramolecular electrostatic interaction with functional domains. In this work, we investigated the impacts of D/E repeats on the target DNA search kinetics for the high-mobility group box 1 (HMGB1) protein and the artificial protein constructs of the Antp homeodomain fused with D/E repeats of varied lengths. Our experimental data showed that D/E repeats of particular lengths can accelerate the target association in the overwhelming presence of non-functional high-affinity ligands ('decoys'). Our coarse-grained molecular dynamics (CGMD) simulations showed that the autoinhibited proteins can bind to DNA and transition into the uninhibited complex with DNA through an electrostatically driven induced-fit process. In conjunction with the CGMD simulations, our kinetic model can explain how D/E repeats can accelerate the target association process in the presence of decoys. This study illuminates an unprecedented role of the negatively charged IDRs in the target search process.

Many DNA/RNA-binding proteins possess intrinsically disordered regions (IDRs) with large negative charge, some of which involve a consecutive sequence of aspartate (D) or glutamate (E) residues. We refer to them as D/E repeats. The functional role of D/E repeats is not well understood, though some of them are known to cause autoinhibition. Here, using the HMGB1 protein and the artificial protein constructs of the Antp homeodomain fused with D/E repeats, we demonstrate that D/E repeats can accelerate the target search process in the presence of non-functional high-affinity ligands ('decoys'). Our coarse-grained molecular dynamics (CGMD) simulations and kinetic model provide mechanistic insight into this acceleration. Our current study illuminates an unprecedented role of the negatively charged IDRs.

Phosphorylation by Protein Kinase C Weakens DNA-Binding Affinity and Folding Stability of the HMGB1 Protein.

The HMGB1 protein typically serves as a DNA chaperone that assists DNA-repair enzymes and transcription factors but can translocate from the nucleus to the cytoplasm or even to extracellular space upon some cellular stimuli. One of the factors that triggers the translocation of HMGB1 is its phosphorylation near a nuclear localization sequence by protein kinase C (PKC), although the exact modification sites on HMGB1 remain ambiguous. In this study, using spectroscopic methods, we investigated the HMGB1 phosphorylation and its impact on the molecular properties of the HMGB1 protein. Our nuclear magnetic resonance (NMR) data on the full-length HMGB1 protein showed that PKC specifically phosphorylates the A-box domain, one of the DNA binding domains of HMGB1. Phosphorylation of S46 and S53 was particularly efficient. Over a longer reaction time, PKC phosphorylated some additional residues within the HMGB1 A-box domain. Our fluorescence-based binding assays showed that the phosphorylation significantly reduces the binding affinity of HMGB1 for DNA. Based on the crystal structures of HMGB1-DNA complexes, this effect can be ascribed to electrostatic repulsion between the negatively charged phosphate groups at the S46 side chain and DNA backbone. Our data also showed that the phosphorylation destabilizes the folding of the A-box domain. Thus, phosphorylation by PKC weakens the DNA-binding affinity and folding stability of HMGB1.

Influence of an Intrinsically Disordered Region on Protein Domains Revealed by NMR-Based Electrostatic Potential Measurements.

Many human proteins possess intrinsically disordered regions containing consecutive aspartate or glutamate residues ("D/E repeats"). Approximately half of them are DNA/RNA-binding proteins. In this study, using nuclear magnetic resonance (NMR) spectroscopy, we investigated the electrostatic properties of D/E repeats and their influence on folded domains within the same protein. Local electrostatic potentials were directly measured for the HMGB1 protein, its isolated D/E repeats, and DNA-binding domains by NMR. The data provide quantitative information about the electrostatic interactions between distinct segments of HMGB1. Due to the interactions between the D/E repeats and the DNA-binding domains, local electrostatic potentials of the DNA-binding domains within the full-length HMGB1 protein were largely negative despite the presence of many positively charged residues. Our NMR data on counterions and electrostatic potentials show that the D/E repeats and DNA have similar electrostatic properties and compete for the DNA-binding domains. The competition promotes dissociation of the protein-DNA complex and influences the molecular behavior of the HMGB1 protein. These effects may be general among the DNA/RNA-binding proteins with D/E repeats.

Robust Enzymatic Production of DNA G-Quadruplex, Aptamer, DNAzyme, and Other Oligonucleotides: Applications for NMR.

Single-stranded DNA (ssDNA) oligonucleotides are widely used in biological research, therapeutics, biotechnology, and nanomachines. Large-scale enzymatic production of ssDNA oligonucleotides forming noncanonical structures has been difficult. Here, we present a simple and robust method named "palindrome-nicking-dependent amplification" (PaNDA) for enzymatic production of a large amount of ssDNA oligonucleotides. It utilizes a strand-displacing DNA polymerase and a nicking enzyme together with input DNA and deoxynucleotide triphosphates at 55 °C. Scaling up of PaNDA is straightforward due to its isothermal nature. The ssDNA products can easily be isolated through anion-exchange chromatography under nondenaturing conditions. We demonstrate applications of PaNDA to 13C/15N-labeling of various DNA strands, including a 22-nt telomere repeat G-quadruplex, a 26-nt therapeutic aptamer, and a 33-nt DNAzyme. The 13C/15N-labeling by PaNDA greatly facilitates the characterization of noncanonical DNA by nuclear magnetic resonance (NMR) spectroscopy. For example, the behavior of therapeutic DNA aptamers in human serum can be investigated.

Analyzing paramagnetic NMR data on target DNA search by proteins using a discrete-state kinetic model for translocation.

Before reaching their targets, sequence-specific DNA-binding proteins nonspecifically bind to DNA through electrostatic interactions and stochastically change their locations on DNA. Investigations into the dynamics of DNA-scanning by proteins are nontrivial due to the simultaneous presence of multiple translocation mechanisms and many sites for the protein to nonspecifically bind to DNA. Nuclear magnetic resonance (NMR) spectroscopy can provide information about the target DNA search processes at an atomic level. Paramagnetic relaxation enhancement (PRE) is particularly useful to study how the proteins scan DNA in the search process. Previously, relatively simple two-state or three-state exchange models were used to explain PRE data reflecting the target search process. In this work, using more realistic discrete-state stochastic kinetics models embedded into an NMR master equation, we analyzed the PRE data for the HoxD9 homeodomain interacting with DNA. The kinetic models that incorporate sliding, dissociation, association, and intersegment transfer can reproduce the PRE profiles observed at some different ionic strengths. The analysis confirms the previous interpretation of the PRE data and shows that the protein's probability distribution among nonspecific sites is nonuniform during the target DNA search process.

DNA base order parameter determination without influence of chemical exchange.

Nuclear magnetic resonance (NMR) spectroscopy is a versatile tool used to investigate the dynamic properties of biological macromolecules and their complexes. NMR relaxation data can provide order parameters S2, which represent the mobility of bond vectors reorienting within a molecular frame. Determination of S2 parameters typically involves the use of transverse NMR relaxation rates. However, the accuracy in S2 determination can be diminished by elevation of the transverse relaxation rates through conformational or chemical exchange involving protonation/deprotonation or non-Watson-Crick base-pair states of nucleic acids. Here, we propose an approach for determination of S2 parameters without the influence of exchange processes. This approach utilizes transverse and longitudinal 13C chemical shift anisotropy (CSA) - dipole-dipole (DD) cross-correlation rates instead of 13C transverse relaxation rates. Anisotropy in rotational diffusion is taken into consideration. An application of this approach to nucleotide base CH groups of a uniformly 13C/15N-labeled DNA duplex is demonstrated.

Quantifying and visualizing weak interactions between anions and proteins.

The molecular properties of proteins are influenced by various ions present in the same solution. While site-specific strong interactions between multivalent metal ions and proteins are well characterized, the behavior of other ions that are only weakly interacting with proteins remains elusive. In the current study, using NMR spectroscopy, we have investigated anion-protein interactions for three proteins that are similar in size but differ in overall charge. Using a unique NMR-based approach, we quantified anions accumulated around the proteins. The determined numbers of anions that are electrostatically attracted to the charged proteins were notably smaller than the overall charge valences and were consistent with predictions from the Poisson-Boltzmann theory. This NMR-based approach also allowed us to measure ionic diffusion and characterize the anions interacting with the positively charged proteins. Our data show that these anions rapidly diffuse while bound to the proteins. Using the same experimental approach, we observed the release of the anions from the protein surface upon the formation of the Antp homeodomain-DNA complex. Using paramagnetic relaxation enhancement (PRE), we visualized the spatial distribution of anions around the free proteins and the Antp homeodomain-DNA complex. The obtained PRE data revealed the localization of anions in the vicinity of the highly positively charged regions of the free Antp homeodomain and provided further evidence of the release of anions from the protein surface upon the protein-DNA association. This study sheds light on the dynamic behavior of anions that electrostatically interact with proteins.

De novo determination of near-surface electrostatic potentials by NMR.

Electrostatic potentials computed from three-dimensional structures of biomolecules by solving the Poisson-Boltzmann equation are widely used in molecular biophysics, structural biology, and medicinal chemistry. Despite the approximate nature of the Poisson-Boltzmann theory, validation of the computed electrostatic potentials around biological macromolecules is rare and methodologically limited. Here, we present a unique and powerful NMR method that allows for straightforward and extensive comparison with electrostatic models for biomolecules and their complexes. This method utilizes paramagnetic relaxation enhancement arising from analogous cationic and anionic cosolutes whose spatial distributions around biological macromolecules reflect electrostatic potentials. We demonstrate that this NMR method enables de novo determination of near-surface electrostatic potentials for individual protein residues without using any structural information. We applied the method to ubiquitin and the Antp homeodomain-DNA complex. The experimental data agreed well with predictions from the Poisson-Boltzmann theory. Thus, our experimental results clearly support the validity of the theory for these systems. However, our experimental study also illuminates certain weaknesses of the Poisson-Boltzmann theory. For example, we found that the theory predicts stronger dependence of near-surface electrostatic potentials on ionic strength than observed in the experiments. Our data also suggest that conformational flexibility or structural uncertainties may cause large errors in theoretical predictions of electrostatic potentials, particularly for highly charged systems. This NMR-based method permits extensive assessment of near-surface electrostatic potentials for various regions around biological macromolecules and thereby may facilitate improvement of the computational approaches for electrostatic potentials.

DNA-mediated proteolysis by neutrophil elastase enhances binding activities of the HMGB1 protein.

Neutrophil extracellular traps (NETs) are produced through ejection of genomic DNA by neutrophils into extracellular space and serve as a weapon to fight against pathogens. Neutrophil elastase, a serine protease loaded on NETs, attacks and kills pathogens, while extracellular high-mobility-group-box-1 (HMGB1) protein serves as a danger signal to other cells. How the action of these factors is coordinated as part of the innate immune response is not fully understood. In this article, using biochemical and biophysical approaches, we demonstrate that DNA mediates specific proteolysis of HMGB1 by neutrophil elastase and that the proteolytic processing remarkably enhances binding activities of extracellular HMGB1. Through the DNA-mediated proteolysis of HMGB1 by neutrophil elastase, the negatively charged segment containing D/E repeats is removed from HMGB1. This proteolytic removal of the C-terminal tail causes a substantial increase in binding activities of HMGB1 because the D/E repeats are crucial for dynamic autoinhibition via electrostatic interactions. Our data on the oxidized HMGB1 (i.e., 'disulfide HMGB1') protein show that the truncation substantially increases HMGB1's affinities for the toll-like receptor TLR4•MD-2 complex, DNA G-quadruplex, and the Holliday junction DNA structure. The DNA-mediated proteolysis of HMGB1 by neutrophil elastase in NETs may promote the function of extracellular HMGB1 as a damage-associated molecular pattern that triggers the innate immune response of nearby cells.

Diffusion NMR-based comparison of electrostatic influences of DNA on various monovalent cations.

Counterions are important constituents for the structure and function of nucleic acids. Using 7Li and 133Cs nuclear magnetic resonance (NMR) spectroscopy, we investigated how ionic radii affect the behavior of counterions around DNA through diffusion measurements of Li+ and Cs+ ions around a 15-bp DNA duplex. Together with our previous data on 23Na+ and 15NH4+ ions around the same DNA under the same conditions, we were able to compare the dynamics of four different monovalent ions around DNA. From the apparent diffusion coefficients at varied concentrations of DNA, we determined the diffusion coefficients of these cations inside and outside the ion atmosphere around DNA (Db and Df, respectively). We also analyzed ionic competition with K+ ions for the ion atmosphere and assessed the relative affinities of these cations for DNA. Interestingly, all cations (i.e., Li+, Na+, NH4+, and Cs+) analyzed by diffusion NMR spectroscopy exhibited nearly identical Db/Df ratios despite the differences in their ionic radii, relative affinities, and diffusion coefficients. These results, along with the theoretical relationship between diffusion and entropy, suggest that the entropy change due to the release of counterions from the ion atmosphere around DNA is also similar regardless of the monovalent ion types. These findings and the experimental diffusion data on the monovalent ions are useful for examination of computational models for electrostatic interactions or ion solvation.

Slow Rotational Dynamics of Cytosine NH2 Groups in Double-Stranded DNA.

Aromatic NH2 groups are essential as hydrogen-bond donors in secondary structures of DNA and RNA. Although rapid rotations of NH2 groups of adenine and guanine bases were previously characterized, there has been a lack of quantitative information about slow rotations of cytosine NH2 groups in Watson-Crick base pairs. In this study, using an NMR method we had recently developed, we determined the kinetic rate constants and energy barriers for cytosine NH2 rotations in a 15-base-pair DNA duplex. Our data show that the rotational dynamics of cytosine NH2 groups depend on local environments. Qualitative correlation between the ranges of 15N chemical shifts and rotational time scales for various NH2 groups of nucleic acids and proteins illuminates a relationship between the partial double-bond character of the C-N bond and the time scale for NH2 rotations.

Dynamics of Cations around DNA and Protein as Revealed by 23Na Diffusion NMR Spectroscopy.

Counterions are vital for the structure and function of biomolecules. However, the behavior of counterions remains elusive due to the difficulty in characterizing mobile ions. Here, we demonstrate that the dynamics of cations around biological macromolecules can be revealed by 23Na diffusion nuclear magnetic resonance (NMR) spectroscopy. NMR probe hardware capable of generating strong magnetic field gradients enables 23Na NMR-based diffusion measurements for Na+ ions in solutions of biological macromolecules and their complexes. The dynamic properties of Na+ ions interacting with the macromolecules can be investigated using apparent 23Na diffusion coefficients measured under various conditions. Our diffusion data clearly show that Na+ ions retain high mobility within the ion atmosphere around DNA. The 23Na diffusion NMR method also permits direct observation of the release of Na+ ions from nucleic acids upon protein-nucleic acid association. The entropy change due to the ion release can be estimated from the diffusion data.

Dynamics of Ionic Interactions at Protein-Nucleic Acid Interfaces.

Molecular association of proteins with nucleic acids is required for many biological processes essential to life. Electrostatic interactions via ion pairs (salt bridges) of nucleic acid phosphates and protein side chains are crucial for proteins to bind to DNA or RNA. Counterions around the macromolecules are also key constituents for the thermodynamics of protein-nucleic acid association. Until recently, there had been only a limited amount of experiment-based information about how ions and ionic moieties behave in biological macromolecular processes. In the past decade, there has been significant progress in quantitative experimental research on ionic interactions with nucleic acids and their complexes with proteins. The highly negatively charged surfaces of DNA and RNA electrostatically attract and condense cations, creating a zone called the ion atmosphere. Recent experimental studies were able to examine and validate theoretical models on ions and their mobility and interactions with macromolecules. The ionic interactions are highly dynamic. The counterions rapidly diffuse within the ion atmosphere. Some of the ions are released from the ion atmosphere when proteins bind to nucleic acids, balancing the charge via intermolecular ion pairs of positively charged side chains and negatively charged backbone phosphates. Previously, the release of counterions had been implicated indirectly by the salt-concentration dependence of the association constant.Recently, direct detection of counterion release by NMR spectroscopy has become possible and enabled more accurate and quantitative analysis of the counterion release and its entropic impact on the thermodynamics of protein-nucleic acid association. Recent studies also revealed the dynamic nature of ion pairs of protein side chains and nucleic acid phosphates. These ion pairs undergo transitions between two major states. In one of the major states, the cation and the anion are in direct contact and form hydrogen bonds. In the other major state, the cation and the anion are separated by water. Transitions between these states rapidly occur on a picosecond to nanosecond time scale. When proteins interact with nucleic acids, interfacial arginine (Arg) and lysine (Lys) side chains exhibit considerably different behaviors. Arg side chains show a higher propensity to form rigid contacts with nucleotide bases, whereas Lys side chains tend to be more mobile at the molecular interfaces. The dynamic ionic interactions may facilitate adaptive molecular recognition and play both thermodynamic and kinetic roles in protein-nucleic acid interactions.

Racemic phosphorothioate as a tool for NMR investigations of protein-DNA complexes.

A major driving force for protein-nucleic acid association is electrostatic interactions via ion pairs of the positively charged basic side chains and negatively charged phosphates. For a better understanding of how proteins scan DNA and recognize particular signatures, it is important to gain atomic-level insight into the behavior of basic side chains at the protein-DNA interfaces. NMR spectroscopy is a powerful tool for investigating the structural, dynamic, and kinetic aspects of protein-DNA interactions. However, resonance assignment of basic side-chain cationic moieties at the molecular interfaces remains to be a major challenge. Here, we propose a fast, robust, and inexpensive approach that greatly facilitates resonance assignment of interfacial moieties and also allows for kinetic measurements of protein translocation between two DNA duplexes. This approach utilizes site-specific incorporation of racemic phosphorothioate at the position of a phosphate that interacts with a protein side chain. This modification retains the electric charge of phosphate and therefore is mild, but causes significant chemical shift perturbations for the proximal protein side chains, which facilitates resonance assignment. Due to the racemic nature of the modification, two different chemical shifts are observed for the species with different diastereomers RP and SP of the incorporated phosphorothioate group. Kinetic information on the exchange of the protein molecule between RP and SP DNA duplexes can be obtained by 15Nz exchange spectroscopy. We demonstrate the applications of this approach to the Antennapedia homeodomain-DNA complex and the CREB1 basic leucine-zipper (bZIP)-DNA complex.

Detecting Counterion Dynamics in DNA-Protein Association.

Due to a high density of negative charges on its surface, DNA condenses cations as counterions, forming the so-called "ion atmosphere". Although the release of counterions upon DNA-protein association has been postulated to have a major contribution to the binding thermodynamics, this release remains to be confirmed through a direct observation of the ions. Herein, we report the characterization of the ion atmosphere around DNA using NMR spectroscopy and directly detect the release of counterions upon DNA-protein association. NMR-based diffusion data reveal the highly dynamic nature of counterions within the ion atmosphere around DNA. Counterion release is observed as an increase in the apparent ionic diffusion coefficient, which directly provides the number of counterions released upon DNA-protein association.

NMR-based investigations into target DNA search processes of proteins.

To perform their function, transcription factors and DNA-repair/modifying enzymes must first locate their targets in the vast presence of nonspecific, but structurally similar sites on genomic DNA. Before reaching their targets, these proteins stochastically scan DNA and dynamically move from one site to another on DNA. Solution NMR spectroscopy provides unique atomic-level insights into the dynamic DNA-scanning processes, which are difficult to gain by any other experimental means. In this review, we provide an introductory overview on the NMR methods for the structural, dynamic, and kinetic investigations of target DNA search by proteins. We also discuss advantages and disadvantages of these NMR methods over other methods such as single-molecule techniques and biochemical approaches.

Potential role of DNA methylation as a facilitator of target search processes for transcription factors through interplay with methyl-CpG-binding proteins.

Eukaryotic genomes contain numerous non-functional high-affinity sequences for transcription factors. These sequences potentially serve as natural decoys that sequester transcription factors. We have previously shown that the presence of sequences similar to the target sequence could substantially impede association of the transcription factor Egr-1 with its targets. In this study, using a stopped-flow fluorescence method, we examined the kinetic impact of DNA methylation of decoys on the search process of the Egr-1 zinc-finger protein. We analyzed its association with an unmethylated target site on fluorescence-labeled DNA in the presence of competitor DNA duplexes, including Egr-1 decoys. DNA methylation of decoys alone did not affect target search kinetics. In the presence of the MeCP2 methyl-CpG-binding domain (MBD), however, DNA methylation of decoys substantially (∼10-30-fold) accelerated the target search process of the Egr-1 zinc-finger protein. This acceleration did not occur when the target was also methylated. These results suggest that when decoys are methylated, MBD proteins can block them and thereby allow Egr-1 to avoid sequestration in non-functional locations. This effect may occur in vivo for DNA methylation outside CpG islands (CGIs) and could facilitate localization of some transcription factors within regulatory CGIs, where DNA methylation is rare.

Impact of two-bond 15N-15N scalar couplings on 15N transverse relaxation measurements for arginine side chains of proteins.

NMR relaxation of arginine (Arg) 15Nε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal 15N-15N scalar couplings on measurements of transverse relaxation rates (R 2 ) for Arg side-chain 15Nε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal 15N-15N couplings ( 2 J NN ) of the 15Nε nuclei and found that the magnitudes of the 2 J NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all 15N nuclei, suggested that the two 2 J NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr-Purcell-Meiboom-Gill (CPMG) scheme for Arg 15Nε R 2 measurements. However, our experimental data show that the expected modulation via two 2 J NN couplings vanishes during the 15N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831-836, 2006). This effect allows for accurate measurements of R 2 relaxation rates for Arg side-chain 15Nε nuclei despite the presence of two 2 J NN couplings. Although the so-called recoupling conditions may cause overestimate of R 2 rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.

Direct detection of lysine side chain NH3+ in protein-heparin complexes using NMR spectroscopy.

Two NMR observables, the NζH3+ peak in the HISQC spectrum and Nζ chemical shift difference between the free and heparin-bound forms, can identify binding-interface lysines in protein-heparin complexes. Unlike backbone chemical shifts, these direct probes are stringent and are less prone to either false positives or false negatives.