Intermolecular and intramolecular readout mechanisms in protein-DNA recognition

Protein-DNA recognition plays an essential role in the regulation of gene expression. Regulatory proteins are known to recognize specific DNA sequences directly through atomic contacts (intermolecular readout) and/or indirectly through the conformational properties of the DNA (intramolecular readout). However, little is known about the respective contributions made by these so-called direct and indirect readout mechanisms. We addressed this question by making use of information extracted from a structural database containing many protein-DNA complexes. We quantified the specificity of intermolecular (direct) readout by statistical analysis of base-amino acid interactions within protein-DNA complexes. The specificity of the intramolecular (indirect) readout due to DNA was quantified by statistical analysis of the sequence-dependent DNA conformation. Systematic comparison of these specificities in a large number of protein-DNA complexes revealed that both intermolecular and intramolecular readouts contribute to the specificity of protein-DNA recognition, and that their relative contributions vary depending upon the protein-DNA complexes. We demonstrated that combination of the intermolecular and intramolecular energies derived from the statistical analyses lead to enhanced specificity, and that the combined energy could explain experimental data on binding affinity changes caused by base mutations. These results provided new insight into the relationship between specificity and structure in the process of protein-DNA recognition, which would lead to prediction of specific protein-DNA binding sites.     

Recognition of 5'-YpG-3' sequences by coupled stacking/hydrogen bonding interactions with amino acid residues

The combined biochemical and structural study of hundreds of protein-DNA complexes has indicated that sequence-specific interactions are mediated by two mechanisms termed direct and indirect readout. Direct readout involves direct interactions between the protein and base-specific atoms exposed in the major and minor grooves of DNA. For indirect readout, the protein recognizes DNA by sensing conformational variations in the structure dependent on nucleotide sequence, typically through interactions with the phosphodiester backbone. Based on our recent structure of Ndt80 bound to DNA in conjunction with a search of the existing PDB database, we propose a new method of sequence-specific recognition that utilizes both direct and indirect readout. In this mode, a single amino acid side-chain recognizes two consecutive base-pairs. The 3'-base is recognized by canonical direct readout, while the 5'-base is recognized through a variation of indirect readout, whereby the conformational flexibility of the particular dinucleotide step, namely a 5'-pyrimidine-purine-3' step, facilitates its recognition by the amino acid via cation-pi interactions. In most cases, this mode of DNA recognition helps explain the sequence specificity of the protein for its target DNA.     

Interpreting protein/DNA interactions: distinguishing specific from non-specific and electrostatic from non-electrostatic components

We discuss the effectiveness of existing methods for understanding the forces driving the formation of specific protein-DNA complexes. Theoretical approaches using the Poisson-Boltzmann (PB) equation to analyse interactions between these highly charged macromolecules to form known structures are contrasted with an empirical approach that analyses the effects of salt on the stability of these complexes and assumes that release of counter-ions associated with the free DNA plays the dominant role in their formation. According to this counter-ion condensation (CC) concept, the salt-dependent part of the Gibbs energy of binding, which is defined as the electrostatic component, is fully entropic and its dependence on the salt concentration represents the number of ionic contacts present in the complex. It is shown that although this electrostatic component provides the majority of the Gibbs energy of complex formation and does not depend on the DNA sequence, the salt-independent part of the Gibbs energy--usually regarded as non-electrostatic--is sequence specific. The CC approach thus has considerable practical value for studying protein/DNA complexes, while practical applications of PB analysis have yet to demonstrate their merit.     

Protein-DNA docking with a coarse-grained force field

ABSTRACT: BACKGROUND: Protein-DNA interactions are important for many cellular processes, however structural knowledge for a large fraction of known and putative complexes is still lacking. Computational docking methods aim at the prediction of complex architecture given detailed structures of its constituents. They are becoming an increasingly important tool in the field of macromolecular assemblies, complementing particularly demanding protein-nucleic acids X ray crystallography and providing means for the refinement and integration of low resolution data coming from rapidly advancing methods such as cryoelectron microscopy. RESULTS: We present a new coarse-grained force field suitable for protein-DNA docking. The force field is an extension of previously developed parameter sets for protein-RNA and protein-protein interactions. The docking is based on potential energy minimization in translational and orientational degrees of freedom of the binding partners. It allows for fast and efficient systematic search for native-like complex geometry without any prior knowledge regarding binding site location. CONCLUSIONS: We find that the force field gives very good results for bound docking. The quality of predictions in the case of unbound docking varies, depending on the level of structural deviation from bound geometries. We analyze the role of specific protein-DNA interactions on force field performance, both with respect to complex structure prediction, and the reproduction of experimental binding affinities. We find that such direct, specific interactions only partially contribute to protein-DNA recognition, indicating an important role of shape complementarity and sequence-dependent DNA internal energy, in line with the concept of indirect protein-DNA readout mechanism.     

Direct visualization of the EcoRII-DNA triple synaptic complex by atomic force microscopy

Interactions between distantly separated DNA regions mediated by specialized proteins lead to the formation of synaptic protein-DNA complexes. This is a ubiquitous phenomenon which is critical in various genetic processes. Although such interactions typically occur between two sites, interactions among three specific DNA regions have been identified, and a corresponding model has been proposed. Atomic force microscopy was used to test this model for the EcoRII restriction enzyme and provide direct visualization and characterization of synaptic protein-DNA complexes involving three DNA binding sites. The complex appeared in the images as a two-loop structure, and the length measurements proved the site specificity of the protein in the complex. The protein volume measurements showed that an EcoRII dimer is the core of the three-site synaptosome. Other complexes were identified and analyzed. The protein volume data showed that the dimeric form of the protein is responsible for the formation of other types of synaptic complexes as well. The applications of these results to the mechanisms of the protein-DNA interactions are discussed.     

Free energy contributions to direct readout of a DNA sequence

The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol(-1)), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol(-1) from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken.     

A new method for evaluating the specificity of indirect readout in protein-DNA recognition

Proteins recognize a specific DNA sequence not only through direct contact (direct readout) with base pairs but also through sequence-dependent conformation and/or flexibility of DNA (indirect readout). However, it is difficult to assess the contribution of indirect readout to the sequence specificity. What is needed is a straightforward method for quantifying its contributions to specificity. Using Bayesian statistics, we derived the probability of a particular sequence for a given DNA structure from the trajectories of molecular dynamics (MD) simulations of DNAs containing all possible tetramer sequences. Then, we quantified the specificity of indirect readout based on the information entropy associated with the probability. We tested this method with known structures of protein-DNA complexes. This method enabled us to correctly predict those regions where experiments suggested the involvement of indirect readout. The results also indicated new regions where the indirect readout mechanism makes major contributions to the recognition. The present method can be used to estimate the contribution of indirect readout without approximations to the distributions in the conformational ensembles of DNA, and would serve as a powerful tool to study the mechanism of protein-DNA recognition.     

Quantitative parameters for amino acid-base interaction: implications for prediction of protein-DNA binding sites.

Inspection of the amino acid-base interactions in protein-DNA complexes is essential to the understanding of specific recognition of DNA target sites by regulatory proteins. The accumulation of information on protein-DNA co-crystals challenges the derivation of quantitative parameters for amino acid-base interaction based on these data. Here we use the coordinates of 53 solved protein-DNA complexes to extract all non-homologous pairs of amino acid-base that are in close contact, including hydrogen bonds and hydrophobic interactions. By comparing the frequency distribution of the different pairs to a theoretical distribution and calculating the log odds, a quantitative measure that expresses the likelihood of interaction for each pair of amino acid-base could be extracted. A score that reflects the compatibility between a protein and its DNA target can be calculated by summing up the individual measures of the pairs of amino acid-base involved in the complex, assuming additivity in their contributions to binding. This score enables ranking of different DNA binding sites given a protein binding site and vice versa and can be used in molecular design protocols. We demonstrate its validity by comparing the predictions using this score with experimental binding results of sequence variants of zif268 zinc fingers and their DNA binding sites.     

Tethered-Hopping Model for Protein-DNA Binding and Unbinding Based on Sox2-Oct1-Hoxb1 Ternary Complex Simulations

The sliding and hopping models encapsulate the essential protein-DNA binding process for binary complex formation and dissociation. However, the effects of a cofactor protein on the protein-DNA binding process that leads to the formation of a ternary complex remain largely unknown. Here we investigate the effect of the cofactor Sox2 on the binding and unbinding of Oct1 with the Hoxb1 control element. We simulate the association of Oct1 with Sox2-Hoxb1 using molecular dynamics simulations, and the dissociation of Oct1 from Sox2-Hoxb1 using steered molecular dynamics simulations, in analogy to a hopping event of Oct1. We compare the kinetic and thermodynamic properties of three model complexes (the wild-type and two mutants) in which the Oct1-DNA base-specific interactions or the Sox2-Oct1 protein-protein interactions are largely abolished. We find that Oct1-DNA base-specific interactions contribute significantly to the total interaction energy of the ternary complex, and that nonspecific Oct1-DNA interactions are sufficient for driving the formation of the protein-DNA interface. The Sox2-Oct1 protein-protein binding interface is largely hydrophobic, with remarkable shape complementarity. This interface promotes the formation of the ternary complex and slows the dissociation of Oct1 from its DNA-binding site. We propose a simple two-step reaction model of protein-DNA binding, called the tethered-hopping model, that explains the importance of the cofactor Sox2 and may apply to similar ternary protein-DNA complexes.     

Methylene Blue Binding to DNA with Alternating AT Base Sequence: Minor Groove Binding is Favored over Intercalation

Abstract

The results presented in this paper on methylene blue (MB) binding to DNA with AT alternating base sequence complement the data obtained in two former modeling studies of MB binding to GC alternating DNA. In the light of the large amount of experimental data for both systems, this theoretical study is focused on a detailed energetic analysis and comparison in order to understand their different behavior. Since experimental high-resolution structures of the complexes are not available, the analysis is based on energy minimized structural models of the complexes in different binding modes. For both sequences, four different intercalation structures and two models for MB binding in the minor and major groove have been proposed. Solvent electrostatic effects were included in the energetic analysis by using electrostatic continuum theory, and the dependence of MB binding on salt concentration was investigated by solving the non-linear Poisson-Boltzmann equation. We find that the relative stability of the different complexes is similar for the two sequences, in agreement with the interpretation of spectroscopic data. Subtle differences, however, are seen in energy decompositions and can be attributed to the change from symmetric 5′-YpR-3′ intercalation to minor groove binding with increasing salt concentration, which is experimentally observed for the AT sequence at lower salt concentration than for the GC sequence. According to our results, this difference is due to the significantly lower non-electrostatic energy for the minor groove complex with AT alternating DNA, whereas the slightly lower binding energy to this sequence is caused by a higher deformation energy of DNA. The energetic data are in agreement with the conclusions derived from different spectroscopic studies and can also be structurally interpreted on the basis of the modeled complexes. The simple static modeling technique and the neglect of entropy terms and of non-electrostatic solute-solvent interactions, which are assumed to be nearly constant for the compared complexes of MB with DNA, seem to be justified by the results.     

Methylene blue binding to DNA with alternating AT base sequence: minor groove binding is favored over intercalation

The results presented in this paper on methylene blue (MB) binding to DNA with AT alternating base sequence complement the data obtained in two former modeling studies of MB binding to GC alternating DNA. In the light of the large amount of experimental data for both systems, this theoretical study is focused on a detailed energetic analysis and comparison in order to understand their different behavior. Since experimental high-resolution structures of the complexes are not available, the analysis is based on energy minimized structural models of the complexes in different binding modes. For both sequences, four different intercalation structures and two models for MB binding in the minor and major groove have been proposed. Solvent electrostatic effects were included in the energetic analysis by using electrostatic continuum theory, and the dependence of MB binding on salt concentration was investigated by solving the non-linear Poisson-Boltzmann equation. We find that the relative stability of the different complexes is similar for the two sequences, in agreement with the interpretation of spectroscopic data. Subtle differences, however, are seen in energy decompositions and can be attributed to the change from symmetric 5'-YpR-3' intercalation to minor groove binding with increasing salt concentration, which is experimentally observed for the AT sequence at lower salt concentration than for the GC sequence. According to our results, this difference is due to the significantly lower non-electrostatic energy for the minor groove complex with AT alternating DNA, whereas the slightly lower binding energy to this sequence is caused by a higher deformation energy of DNA. The energetic data are in agreement with the conclusions derived from different spectroscopic studies and can also be structurally interpreted on the basis of the modeled complexes. The simple static modeling technique and the neglect of entropy terms and of non-electrostatic solute-solvent interactions, which are assumed to be nearly constant for the compared complexes of MB with DNA, seem to be justified by the results.     

Influence of DNA stiffness in protein-DNA recognition

Protein-DNA recognition plays an essential role in the regulation of gene expression. The protein-DNA binding specificity is based on direct atomic contacts between protein and DNA and/or the conformational properties of DNA. In this work, we have analyzed the influence of DNA stiffness (E) to the specificity of protein-DNA complexes. The average DNA stiffness parameters for several protein-DNA complexes have been computed using the structure based sequence dependent stiffness scale. The relationship between DNA stiffness and experimental protein-DNA binding specificity has been brought out. We have investigated the importance of DNA stiffness with the aid of experimental free energy changes (DeltaDeltaG) due to binding in several protein-DNA complexes, such as, ETS proteins, 434, lambda, Mnt and trp repressors, 434 cro protein, EcoRV endonuclease V and zinc fingers. We found a correlation in the range 0.65-0.97 between DeltaDeltaG and E in these examples. Further, we have qualitatively analyzed the effect of mutations in the target sequence of lambda repressor and we observed that the DNA stiffness could correctly identify 70% of the correct bases among the considered nine positions.