Protein-nucleic acid recognition: statistical analysis of atomic interactions and influence of DNA structure

We analyzed structural features of 11,038 direct atomic contacts (either electrostatic, H-bonds, hydrophobic, or other van der Waals interactions) extracted from 139 protein-DNA and 49 protein-RNA nonhomologous complexes from the Protein Data Bank (PDB). Globally, H-bonds are the most frequent interactions (approximately 50%), followed by van der Waals, hydrophobic, and electrostatic interactions. From the protein viewpoint, hydrophilic amino acids are over-represented in the interaction databases: Positively charged amino acids mainly contact nucleic acid phosphate groups but can also interact with base edges. From the nucleotide point of view, DNA and RNA behave differently: Most protein-DNA interactions involve phosphate atoms, while protein-RNA interactions involve more frequently base edge and ribose atoms. The increased participation of DNA phosphate involves H-bonds rather than salt bridges. A statistical analysis was performed to find the occurrence of amino acid-nucleotide pairs most different from chance. These pairs were analyzed individually. Finally, we studied the conformation of DNA in the interaction sites. Despite the prevalence of B-DNA in the database, our results suggest that A-DNA is favored in the interaction sites.     

Conformational plasticity of RNA for target recognition as revealed by the 2.15?? crystal structure of a human IgG–aptamer complex

Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are applicable to therapeutics and diagnostics. Regardless of an increasing number of reported aptamers, the structural basis of the interaction of RNA aptamer with proteins is poorly understood. Here, we determined the 2.15 Å crystal structure of the Fc fragment of human IgG1 (hFc1) complexed with an anti-Fc RNA aptamer. The aptamer adopts a characteristic structure fit to hFc1 that is stabilized by a calcium ion, and the binding activity of the aptamer can be controlled many times by calcium chelation and addition. Importantly, the aptamer–hFc1 interaction involves mainly van der Waals contacts and hydrogen bonds rather than electrostatic forces, in contrast to other known aptamer–protein complexes. Moreover, the aptamer–hFc1 interaction involves human IgG-specific amino acids, rendering the aptamer specific to human IgGs, and not crossreactive to other species IgGs. Hence, the aptamer is a potent alternative for protein A affinity purification of Fc-fusion proteins and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids.     

Contribution of enthalpy to the energetics of complex formation of aromatic ligands with DNA

The energy contributions of electrostatic, van der Waals interactions, hydrogen bonds, and interactions of charge transfer type to the enthalpy of complex formation of the double-stand DNA with the antitumor antibiotics daunomycin, nogalamycin, and novantron, as well as the mutagens ethidium bromide and proflavine have been calculated. According to the calculations, the van der Waals component (except for nogalamycin) is energetically favorable during complex formation of the antibiotics with DNA, and the contributions of H bonds and electrostatic interactions are unfavorable, with the probability of charge transfer in the complexes being low. It has been shown that the relatively low value of the experimental enthalpy of binding is the sum of components greater in absolute value and different in the sign, which is the cause of large errors in estimating the total enthalpy of complex formation of aromatic ligands with DNA.     

Molecular mechanical perspective on halogen bonding

The nature and strength of halogen bonding in halo molecule-Lewis base complexes were studied in terms of molecular mechanics using our recently developed positive extra-point (PEP) approach, in which the σ-hole on the halogen atom is represented by an extra point of positive charge. The contributions of the σ-hole (i.e., positively charged extra point) and the halogen atom to the strength of this noncovalent interaction were clarified using the atomic parameter contribution to the molecular interaction (APCtMI) approach. The molecular mechanical results revealed that the halogen bond is electrostatic and van der Waals in nature, and its strength depends on three types of interaction: (1) the attractive electrostatic interaction between the σ-hole and the Lewis base, (2) the repulsive electrostatic interaction between the negative halogen atom and the Lewis base, and (3) the repulsive/attractive van der Waals interactions between the halogen atom and the Lewis base. The strength of the halogen bond increases with increasing σ-hole size (i.e., magnitude of the extra-point charge) and increasing halogen atom size. The van der Waals interaction's contribution to the halogen bond strength is most favorable in chloro complexes, whereas the electrostatic interaction is dominant in iodo complexes. The idea that the chloromethane molecule can form a halogen bond with a Lewis base was revisited in terms of quantum mechanics and molecular mechanics. Although chloromethane does produce a positive region along the C-Cl axis, basis set superposition error corrected second-order M?ller-Plesset calculations showed that chloromethane-Lewis base complexes are unstable, producing halogen-Lewis base contacts longer than the sum of the van der Waals radii of the halogen and O/N atoms. Molecular mechanics using the APCtMI approach showed that electrostatic interactions between chloromethane and a Lewis base are unfavorable owing to the high negative charge on the chlorine atom, which overcomes the corresponding favorable van der Waals interactions.     

From cisplatin to artificial nucleases — the role of metal ion-nucleic acid interactions in biology

Metal ions and metal coordination compounds bind to nucleic acids in a variety of ways, ranging from weak electrostatic interactions via hydrogen bonding and/or van der Waals forces to strong covalent binding. Metal ions naturally take part in the formation and the degradation of nucleic acids, and the propensity of certain metal coordination compounds to bind to nucleic acids, notably DNA, is enploited in cancer chemotherapy. Moreover, metal compounds have a wide potential as chemical probes for nucleic acid structures and as tools for nucleic acid processing.     

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein–RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein–double-stranded DNA and protein–single-stranded DNA complexes. The interface properties of the protein–RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein–RNA and protein–DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein–RNA complexes, backbone contacts were more dominant in the protein–DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.     

Study of protein-protein interaction using conformational space annealing

We apply conformational space annealing (CSA), an efficient global optimization method, to the study of protein-protein interaction. The CSA is incorporated into the Tinker molecular modeling package along with a B-spline method for CAPRI Round 5 experiments. We have used an energy function for the protein-protein interaction that consists of electrostatic interaction, van der Waals interaction, and solvation energy terms represented by the occupancy desolvation method. The parameters of the AMBER94 all-atom empirical force field are used. Each energy term is calculated by precalculated grid potentials and B-spline method approximation. The ligand protein is placed inside a sphere of 50 A radius centered at an appropriate location, and the CSA rigid docking studies are carried out to find stable complexes. Up to 10 complexes are selected using the K-mean clustering method and biological information when available. These complexes are energy-minimized for further refinement by considering the flexibility of interacting proteins. The results show that the CSA method has a potential for the study of protein-protein interaction.     

Ethidium bromide-dinucleotide complexes. Evidence for intercalation and sequence preferences in binding to double-stranded nucleic acids.

The solution complexes of ethidium bromide with nine different deoxydinucleotides and the four self-complementary ribodinucleoside monophosphates as well as mixtures of complementary and noncomplementary deoxydinucleotides were studied as models for the binding of the drug to DNA and RNA. Ethidium bromide forms the strongest complexes with pdC-dG and CpG and shows a definite preference for interaction with pyrimidine–purine sequence isomers. Cooperativity is observed in the binding curves of the self-complementary deoxydinucleotides pdC-dG and pdG-dC as well as the ribodinucleoside monophosphates CpG and GpC, indicating the formation of a minihelix around ethidium bromide. The role of complementarity of the nucleotide bases was evident in the visible and circular dichroism spectra of mixtures of complementary and noncomplementary dinucleotides. Nuclear magnetic resonance measurements on an ethidium bromide complex with CpG provided evidence for the intercalation model for the binding of ethidium bromide to double-stranded nucleic acids. The results also suggest that ethidium bromide may bind to various sequences on DNA and RNA with significantly different binding constants.     

Comparative binding energy analysis of the substrate specificity of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10

Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloroacetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93% (74% cross-validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis.     

Hydrophobicity at the surface of proteins

A new method is presented to quantitatively estimate and graphically display the propensity of nonpolar groups to bind at the surface of proteins. It is based on the calculation of the binding energy, i.e., van der Waals interaction plus protein electrostatic desolvation, of a nonpolar probe sphere rolled over the protein surface, and on the color coding of this quantity on a smooth molecular surface (hydrophobicity map). The method is validated on ten protein-ligand complexes and is shown to distinguish precisely where polar and nonpolar groups preferentially bind. Comparisons with existing approaches, like the display of the electrostatic potential or the curvature, illustrate the advantages and the better predictive power of the present method. Hydrophobicity maps will play an important role in the characterization of binding sites for the large number of proteins emerging from the genome projects and structure modeling approaches.     

Protein-RNA interactions: structural analysis and functional classes

A data set of 89 protein-RNA complexes has been extracted from the Protein Data Bank, and the nucleic acid recognition sites characterized through direct contacts, accessible surface area, and secondary structure motifs. The differences between RNA recognition sites that bind to RNAs in functional classes has also been analyzed. Analysis of the complete data set revealed that van der Waals interactions are more numerous than hydrogen bonds and the contacts made to the nucleic acid backbone occur more frequently than specific contacts to nucleotide bases. Of the base-specific contacts that were observed, contacts to guanine and adenine occurred most frequently. The most favored amino acid-nucleotide pairings observed were lysine-phosphate, tyrosine-uracil, arginine-phosphate, phenylalanine-adenine and tryptophan-guanine. The amino acid propensities showed that positively charged and polar residues were favored as expected, but also so were tryptophan and glycine. The propensities calculated for the functional classes showed trends similar to those observed for the complete data set. However, the analysis of hydrogen bond and van der Waal contacts showed that in general proteins complexed with messenger RNA, transfer RNA and viral RNA have more base specific contacts and less backbone contacts than expected, while proteins complexed with ribosomal RNA have less base-specific contacts than the expected. Hence, whilst the types of amino acids involved in the interfaces are similar, the distribution of specific contacts is dependent upon the functional class of the RNA bound.     

Protein docking using continuum electrostatics and geometric fit

The computer program DOT quickly finds low-energy docked structures for two proteins by performing a systematic search over six degrees of freedom. A novel feature of DOT is its energy function, which is the sum of both a Poisson-Boltzmann electrostatic energy and a van der Waals energy, each represented as a grid-based correlation function. DOT evaluates the energy of interaction for many orientations of the moving molecule and maintains separate lists scored by either the electrostatic energy, the van der Waals energy or the composite sum of both. The free energy is obtained by summing the Boltzmann factor over all rotations at each grid point. Three important findings are presented. First, for a wide variety of protein-protein interactions, the composite-energy function is shown to produce larger clusters of correct answers than found by scoring with either van der Waals energy (geometric fit) or electrostatic energy alone. Second, free-energy clusters are demonstrated to be indicators of binding sites. Third, the contributions of electrostatic and attractive van der Waals energies to the total energy term appropriately reflect the nature of the various types of protein-protein interactions studied.     

Do electrostatic interactions destabilize protein–nucleic acid binding?

The negatively charged phosphates of nucleic acids are often paired with positively charged residues upon binding proteins. It was thus counter-intuitive when previous Poisson-Boltzmann (PB) calculations gave positive energies from electrostatic interactions, meaning that they destabilize protein-nucleic acid binding. Our own PB calculations on protein-protein binding have shown that the sign and the magnitude of the electrostatic component are sensitive to the specification of the dielectric boundary in PB calculations. A popular choice for the boundary between the solute low dielectric and the solvent high dielectric is the molecular surface; an alternative is the van der Waals (vdW) surface. In line with results for protein-protein binding, in this article, we found that PB calculations with the molecular surface gave positive electrostatic interaction energies for two protein-RNA complexes, but the signs are reversed when the vdW surface was used. Therefore, whether destabilizing or stabilizing effects are predicted depends on the choice of the dielectric boundary. The two calculation protocols, however, yielded similar salt effects on the binding affinity. Effects of charge mutations differentiated the two calculation protocols; PB calculations with the vdW surface had smaller deviations overall from experimental data.     

The DNA and RNA specificity of eilatin Ru(II) complexes as compared to eilatin and ethidium bromide

Eilatin-containing ruthenium complexes bind to a broad range of different nucleic acids including: calf thymus (CT) DNA, tRNA(Phe), polymeric RNAs and DNAs, and viral RNAs including the HIV-1 RRE and TAR. The nucleic acid specificity of Lambda- and Delta-[Ru(bpy)2eilatin]2+ have been compared to that of the 'free' eilatin ligand, and to the classic intercalating agent ethidium bromide. Interestingly, all four compounds appear to bind to nucleic acids by intercalation, but the trends in nucleic acid binding specificity are highly diverse. Unlike ethidium bromide, both eilatin and the eilatin-containing coordination complexes bind to certain single-stranded RNAs with high affinity (K(d) < or = 1 microM). Eilatin itself is selective for electron-poor polymeric purines, while the eilatin-coordination complexes exhibit preference for the polypyrimidine r(U). These results show how the binding specificity of an intercalating ligand can change upon its incorporation into an octahedral metal complex.     

PRI-Modeler: extracting RNA structural elements from PDB files of protein-RNA complexes

A complete understanding of protein and RNA structures and their interactions is important for determining the binding sites in protein-RNA complexes. Computational approaches exist for identifying secondary structural elements in proteins from atomic coordinates. However, similar methods have not been developed for RNA, due in part to the very limited structural data so far available. We have developed a set of algorithms for extracting and visualizing secondary and tertiary structures of RNA and for analyzing protein-RNA complexes. These algorithms have been implemented in a web-based program called PRI-Modeler (protein-RNA interaction modeler). Given one or more protein data bank files of protein-RNA complexes, PRI-Modeler analyzes the conformation of the RNA, calculates the hydrogen bond (H bond) and van der Waals interactions between amino acids and nucleotides, extracts secondary and tertiary RNA structure elements, and identifies the patterns of interactions between the proteins and RNAs. This paper presents PRI-Modeler and its application to the hydrogen bond and van der Waals interactions in the most representative set of protein-RNA complexes. The analysis reveals several interesting interaction patterns at various levels. The information provided by PRI-Modeler should prove useful for determining the binding sites in protein-RNA complexes. PRI-Modeler is accessible at http://wilab.inha.ac.kr/primodeler/, and supplementary materials are available in the analysis results section at http://wilab.inha.ac.kr/primodeler/.     

A free energy analysis of nucleic acid base stacking in aqueous solution.

This paper reports a theoretical study of the free energy contributions to nucleic acid base stacking in aqueous solution. Electrostatic interactions are treated by using the finite difference Poisson-Boltzmann method and nonpolar effects are treated with explicit calculation of van der Waals interactions and/or free energy-surface area relationships. Although for some pairs of bases there is a favorable Coulombic interaction in the stacked conformation, generally the net effect of electrostatic interactions is to oppose stacking. This result is caused by the loss of favorable base-solvent electrostatic interactions, that accompany the partial removal of polar atoms from water in the stacked conformation. Nonpolar interactions, involving the hydrophobic effect and enhancement of van der Waals interactions caused by close-packing, drive stacking. The calculations qualitatively reproduce the experimental dependence of stacking free energy on purine-pyrimidine composition.     

Direct measurements of forces between phosphatidylcholine and phosphatidylethanolamine bilayers in aqueous electrolyte solutions

We report direct measurements of the full interbilayer force laws (force vs. distance) between bilayers of various phosphatidylcholines and phosphatidylethanolamine in aqueous solutions. Bilayers were first deposited on molecularly smooth (mica) surfaces and the interbilayer forces then measured at a resolution of 1 A. Three types of forces were identified: attractive van der Waals forces, repulsive electrostatic (double-layer) forces, and (at short range) repulsive steric hydration forces. Double-layer forces, which arise from ion binding, were insignificant in monovalent salt solutions, e.g., NaCl up to 1 M, but were already present in solutions containing millimolar levels of CaCl2 and MgCl2, giving rise to forces in excellent agreement with theory. Ca2+ binds more strongly than Mg2+, and both bind less to lecithin bilayers in the fluid state (T greater than Tc). The plane of charge coincides with the location of the negative phosphate groups, while the effective plane of origin of the van der Waals force is 4-5 A farther out. In water, the adhesion energies are in the range 0.10-0.15 erg/cm2 for lecithins and approximately 0.8 erg/cm2 for phosphatidylethanolamine. The adhesion energies vary on addition of salt due to changes in the repulsive double-layer and hydration forces rather than to a change in the attractive van der Waals force. The short-range repulsive forces which balance the van der Waals force at separations of 10-30 A are due to a combination of hydration and steric repulsions, the latter arising from thermal motions of head groups and thickness fluctuations of fluid bilayers (above Tc). It is also concluded that bilayer fusion is not simply related to the interbilayer force law.     

Electrostatic contribution to the binding stability of protein-protein complexes

To investigate roles of electrostatic interactions in protein binding stability, electrostatic calculations were carried out on a set of 64 mutations over six protein-protein complexes. These mutations alter polar interactions across the interface and were selected for putative dominance of electrostatic contributions to the binding stability. Three protocols of implementing the Poisson-Boltzmann model were tested. In vdW4 the dielectric boundary between the protein low dielectric and the solvent high dielectric is defined as the protein van der Waals surface and the protein dielectric constant is set to 4. In SE4 and SE20, the dielectric boundary is defined as the surface of the protein interior inaccessible to a 1.4-A solvent probe, and the protein dielectric constant is set to 4 and 20, respectively. In line with earlier studies on the barnase-barstar complex, the vdW4 results on the large set of mutations showed the closest agreement with experimental data. The agreement between vdW4 and experiment supports the contention of dominant electrostatic contributions for the mutations, but their differences also suggest van der Waals and hydrophobic contributions. The results presented here will serve as a guide for future refinement in electrostatic calculation and inclusion of nonelectrostatic effects.     

The nature of intermolecular interactions between aromatic amino acid residues

The nature of intermolecular interactions between aromatic amino acid residues has been investigated by a combination of molecular dynamics and ab initio methods. The potential energy surface of various interacting pairs, including tryptophan, phenilalanine, and tyrosine, was scanned for determining all the relevant local minima by a combined molecular dynamics and conjugate gradient methodology with the AMBER force field. For each of these minima, single-point correlated ab initio calculations of the binding energy were performed. The agreement between empirical force field and ab initio binding energies of the minimum energy structures is excellent. Aromatic-aromatic interactions can be rationalized on the basis of electrostatic and van der Waals interactions, whereas charge transfer or polarization phenomena are small for all intermolecular complexes and, particularly, for stacked structures. Proteins 2002;48:117-125.     

Stereochemical complementarity of progesterone, RU486 and cavities between base pairs in partially unwound double stranded DNA assessed by computer modeling and energy calculations

Computer modeling was used to examine the relative fit of progesterone and RU486 in cavities constructed between base pairs in double stranded DNA. Progesterone was capable of forming two stereospecific hydrogen bonds between the carbonyl groups and protonated phosphate groups on adjacent strands. Favorable van der Waals and electrostatic energies were exhibited upon insertion of progesterone into DNA indicating an excellent fit. While RU486 could be accommodated between the base pairs and formed hydrogen bonds, there was a high van der Waals energy in the resulting complex. When the complexes were subjected to energy minimization, the conformation of the DNA was significantly altered in the RU486-DNA complex but not in the progesterone-DNA complex. No mechanistic interpretation of these results is proffered; however, such information may have evolutionary significance and could prove useful in designing new progesterone agonists and antagonists.