Protein-RNA contacts at crystal packing surfaces

The crystal packing surfaces comprising protein-RNA interactions were analyzed for 50 RNA-protein crystal structures in the Protein Data Bank database. Protein-RNA crystal contacts, which represent nonspecific protein-RNA interfaces, were investigated for their amino acid propensities, hydrogen bond patterns, and backbone and side chain interactions. When compared to biologically relevant interactions, the protein-RNA crystal contacts exhibit similarities as well as differences with respect to the principles of protein-RNA interactions. Similar to what was observed at cognate protein-RNA interfaces, positively charged amino acids have high propensities at noncognate protein-RNA interfaces and preferentially form hydrogen bonds with RNA phosphate groups. In contrast, nonpolar residues are less frequently associated with noncognate interactions. These results highlight the important roles of both electrostatic and hydrogen bonding interactions, facilitated by positively charged amino acids, in mediating both specific and nonspecific protein-RNA interactions.     

Statistical analysis of atomic contacts at RNA-protein interfaces

Forty-five crystals of complexes between proteins and RNA molecules from the Protein Data Bank have been statistically surveyed for the number of contacts between RNA components (phosphate, ribose and the four bases) and amino acid side chains. Three groups of complexes were defined: the tRNA synthetases; the ribosomal complexes; and a third group containing a variety of complexes. The types of atomic contacts were a priori classified into ionic, neutral H-bond, C-H...O H-bond, or van der Waals interaction. All the contacts were organized into a relational database which allows for statistical analysis. The main conclusions are the following: (i) in all three groups of complexes, the most preferred amino acids (Arg, Asn, Ser, Lys) and the less preferred ones (Ala, Ile, Leu, Val) are the same; Trp and Cys are rarely observed (respectively 15 and 5 amino acids in the ensemble of interfaces); (ii) of the total number of amino acids located at the interfaces 22% are hydrophobic, 40% charged (positive 32%, negative 8%), 30% polar and 8% are Gly; (iii) in ribosomal complexes, phosphate is preferred over ribose, which is preferred over the bases, but there is no significant preference in the other two groups; (iv) there is no significant prevalence of a base type at protein-RNA interfaces, but specifically Arg and Lys display a preference for phosphate over ribose and bases; Pro and Asn prefer bases over ribose and phosphate; Met, Phe and Tyr prefer ribose over phosphate and bases. Further, Ile, Pro, Ser prefer A over the others; Leu prefers C; Asp and Gly prefer G; and Asn prefers U. Considering the contact types, the following conclusions could be drawn: (i) 23% of the contacts are via potential H-bonds (including CH...O H-bonds and ionic interactions), 72% belong to van der Waals interactions and 5% are considered as short contacts; (ii) of all potential H-bonds, 54% are standard, 33% are of the C-H...O type and 13% are ionic; (iii) the Watson-Crick sites of G, O6(G) and principally N2(G) and the hydroxyl group O2' is more often involved in H-bonds than expected; the protein main chain is involved in 32% and the side chains in 68% of the H-bonds; considering the neutral and ionic H-bonds, the following couples are more frequent than expected-base A-Ser, base G-Asp/Glu, base U-Asn. The RNA CH groups interact preferentially with oxygen atoms (62% on the main chain and 19% on the side chains); (iv) the bases are involved in 38% of all H-bonds and more than 26% of the H-bonds have the H donor group on the RNA; (v) the atom O2' is involved in 21% of all H-bonds, a number greater than expected; (vi) amino acids less frequently in direct contact with RNA components interact frequently via their main chain atoms through water molecules with RNA atoms; in contrast, those frequently observed in direct contact, except Ser, use instead their side chain atoms for water bridging interactions.     

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.     

Nonsequence-specific arginine interactions in the nucleosome core particle

We have analyzed the relative orientation of basic amino acid side chains towards DNA in the nucleosome core particle. The electric field created by DNA phosphates has no apparent preferential orientation: no favored orientation of the arginine guanidinium group is found. Arginine may be either directly hydrogen bonded to a phosphate oxygen or stabilized in the minor groove by van der Waals contacts and the local negative electric field. On the other hand, the phosphate oxygen atoms hydrogen bonded to arginines are always found close to the plane defined by the guanidinium group. Thus it can be concluded that the interactions of arginine are strongly directional, those of phosphate are not. We also find that a highly charged fragment of histone H2B, which is placed between two DNA turns, has a very variable conformation. An increase in protein positive charge density apparently allows multiple nonspecific protein conformations when interacting with DNA.     

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.     

Molecular Dynamics Simulation in Solvent of the Estrogen Receptor Protein DNA Binding Domain in Complex with a Non-Consensus Estrogen Response Element DNA Sequence

Abstract

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the estrogen receptor (ER) protein DNA binding domain (DBD) amino acids and DNA of a non-consensus estrogen response element (ERE) consisting of 29 nucleotide base pairs. This ERE nucleotide sequence occurs naturally upstream of the Xenopus laevis Vitelligenin AI gene. The ER DBD is encoded by three exons. Namely, exons 2 and 3 which encode the two zinc binding motifs and a sequence of exon 4 which encodes a predicted alpha helix. We generated a computer model of the ER DBD using atomic coordinates derived from the average of 30 nuclear magnetic resonance (NMR) spectroscopy coordinate sets. Amino acids on the carboxyl end of the ER DBD were disordered in both X-ray crystallography and NMR determinations and no coordinates were reported. This disordered region includes 10 amino acids of a predicted alpha helix encoded in exon 4 at the exon 3/4 splice junction. These amino acids are known to be important in DNA binding and are also believed to function as a nuclear translocation signal sequence for the ER protein. We generated a computer model of the predicted alpha helix consisting of the 10 amino acids encoded in exon 4 and attached this helix to the carboxyl end of the ER DBD at the exon 3/4 splice junction site. We docked the ER DBD model within the DNA major groove halfsites of the 29 base pair non-consensus ERE and flanking nucleotides. We constructed a solvated model with the ER DBD/ERE complex surrounded by a ten Angstrom water layer and conducted molecular dynamics simulations. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the ER DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ERE DNA right major groove halfsite. Amino acids of the ER DBD exon 4 encoded predicted alpha helix formed direct and water mediated H-bonds with base and backbone sites of their cognate codon-anticodon nucleotides within the minor grooves flanking the ERE DNA major groove halfsites. These interactions together induced bending of the DNA into the protein.     

Molecular architecture of protein-RNA recognition sites

The molecular architecture of protein-RNA interfaces are analyzed using a non-redundant dataset of 152 protein-RNA complexes. We find that an average protein-RNA interface is smaller than an average protein-DNA interface but larger than an average protein–protein interface. Among the different classes of protein-RNA complexes, interfaces with tRNA are the largest, while the interfaces with the single-stranded RNA are the smallest. Significantly, RNA contributes more to the interface area than its partner protein. Moreover, unlike protein–protein interfaces where the side chain contributes less to the interface area compared to the main chain, the main chain and side chain contributions flipped in protein-RNA interfaces. We find that the protein surface in contact with the RNA in protein-RNA complexes is better packed than that in contact with the DNA in protein-DNA complexes, but loosely packed than that in contact with the protein in protein–protein complexes. Shape complementarity and electrostatic potential are the two major factors that determine the specificity of the protein-RNA interaction. We find that the H-bond density at the protein-RNA interfaces is similar with that of protein-DNA interfaces but higher than the protein–protein interfaces. Unlike protein-DNA interfaces where the deoxyribose has little role in intermolecular H-bonds, due to the presence of an oxygen atom at the 2′ position, the ribose in RNA plays significant role in protein-RNA H-bonds. We find that besides H-bonds, salt bridges and stacking interactions also play significant role in stabilizing protein-nucleic acids interfaces; however, their contribution at the protein–protein interfaces is insignificant.     

Structure-based analysis of protein-RNA interactions using the program ENTANGLE

Until recently, drawing general conclusions about RNA recognition by proteins has been hindered by the paucity of high-resolution structures. We have analyzed 45 PDB entries of protein-RNA complexes to explore the underlying chemical principles governing both specific and non-sequence specific binding. To facilitate the analysis, we have constructed a database of interactions using ENTANGLE, a JAVA-based program that uses available structural models in their PDB format and searches for appropriate hydrogen bonding, stacking, electrostatic, hydrophobic and van der Waals interactions. The resulting database of interactions reveals correlations that suggest the basis for the discrimination of RNA from DNA and for base-specific recognition. The data illustrate both major and minor interaction strategies employed by families of proteins such as tRNA synthetases, ribosomal proteins, or RNA recognition motifs with their RNA targets. Perhaps most surprisingly, specific RNA recognition appears to be mediated largely by interactions of amide and carbonyl groups in the protein backbone with the edge of the RNA base. In cases where a base accepts a proton, the dominant amino acid donor is arginine, whereas in cases where the base donates a proton, the predominant acceptor is the backbone carbonyl group, not a side-chain group. This is in marked contrast to DNA-protein interactions, which are governed predominantly by amino acid side-chain interactions with functional groups that are presented in the accessible major groove. RNA recognition often proceeds through loops, bulges, kinks and other irregular structures that permit use of all the RNA functional groups and this is seen throughout the protein-RNA interaction database.     

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.     

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/.     

阳离子-π相互作用在两种典型蛋白质折叠型中的偏好性

阳离子-π相互作用是一种在阳离子和芳香性体系之间形成的一种作用力。在蛋白质中,带正电荷的氨基酸(Lys、Arg)和芳香族氨基酸(Phe、Tyr、Trp)之间可以形成阳离子-π相互作用。对α/β类蛋白中两种典型折叠类型――单绕和双绕的研究表明:(1)单绕结构中阳离子-π相互作用的分布密度是双绕结构的2.3倍。(2)Arg-Phe组合偏好在双绕中出现,Arg-Tyr组合偏好在单绕中出现。(3)在单绕中除Lys-Phe组合外,其余5种组合的阳离子-π相互作用能量高于双绕的对应组合,其中以Arg-Trp组合的能量最高。(4)在单绕结构中,样本所含氨基酸残基数量和样本中阳离子-π的数量有明显的相关性,在双绕结构中没有发现类似的相关性。(5)在单绕和双绕结构当中,把阳离子-π相互作用能量分解为静电能和范德华力能揭示出静电能与范德华力能之比接近2∶1,静电作用在阳离子-π相互作用中起主要作用。     

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.     

Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer

We determined the 2.45 A crystal structure of the nucleosome core particle from Drosophila melanogaster and compared it to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human alpha-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein-DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the volume of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged.     

Investigation of Atomic Level Patterns in Protein—Small Ligand Interactions

Background

Shape complementarity and non-covalent interactions are believed to drive protein-ligand interaction. To date protein-protein, protein-DNA, and protein-RNA interactions were systematically investigated, which is in contrast to interactions with small ligands. We investigate the role of covalent and non-covalent bonds in protein-small ligand interactions using a comprehensive dataset of 2,320 complexes.

Methodology and Principal Findings

We show that protein-ligand interactions are governed by different forces for different ligand types, i.e., protein-organic compound interactions are governed by hydrogen bonds, van der Waals contacts, and covalent bonds; protein-metal ion interactions are dominated by electrostatic force and coordination bonds; protein-anion interactions are established with electrostatic force, hydrogen bonds, and van der Waals contacts; and protein-inorganic cluster interactions are driven by coordination bonds. We extracted several frequently occurring atomic-level patterns concerning these interactions. For instance, 73% of investigated covalent bonds were summarized with just three patterns in which bonds are formed between thiol of Cys and carbon or sulfur atoms of ligands, and nitrogen of Lys and carbon of ligands. Similar patterns were found for the coordination bonds. Hydrogen bonds occur in 67% of protein-organic compound complexes and 66% of them are formed between NH- group of protein residues and oxygen atom of ligands. We quantify relative abundance of specific interaction types and discuss their characteristic features. The extracted protein-organic compound patterns are shown to complement and improve a geometric approach for prediction of binding sites.

Conclusions and Significance

We show that for a given type (group) of ligands and type of the interaction force, majority of protein-ligand interactions are repetitive and could be summarized with several simple atomic-level patterns. We summarize and analyze 10 frequently occurring interaction patterns that cover 56% of all considered complexes and we show a practical application for the patterns that concerns interactions with organic compounds.     

The molecular structure of the complex of Hoechst 33258 and the DNA dodecamer d(CGCGAATTCGCG).

The crystal structure of the complex between the dodecamer d(CGCGAATTCGCG) and a synthetic dye molecule Hoechst 33258 was solved by X-ray diffraction analysis and refined to an R-factor of 15.7% at 2.25 A resolution. The crescent-shaped Hoechst compound is found to bind to the central four AATT base pairs in the narrow minor groove of the B-DNA double helix. The piperazine ring of the drug has its flat face almost parallel to the aromatic bisbenzimidazole ring and lies sideways in the minor groove. No evidence of disordered structure of the drug is seen in the complex. The binding of Hoechst to DNA is stabilized by a combination of hydrogen bonding, van der Waals interaction and electrostatic interactions. The binding preference for AT base pairs by the drug is the result of the close contact between the Hoechst molecule and the C2 hydrogen atoms of adenine. The nature of these contacts precludes the binding of the drug to G-C base pairs due to the presence of N2 amino groups of guanines. The present crystal structural information agrees well with the data obtained from chemical footprinting experiments.     

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.     

Molecular dynamic simulation and DFT study on the Drug-DNA interaction; Crocetin as an anti-cancer and DNA nanostructure model

In this research, the interaction of Crocetin as an anti-cancer drug and a Dickerson DNA has been investigated. 25 ns molecular dynamic simulations of Crocetin and DNA composed of 12 base pairs and a sequence of d(CGCGAATTCGCG)₂ were done in water. Three definite parts of the B-DNA were considered in analyzing the best interactive site from the thermodynamic point of view. Binding energy analysis showed that van der Waals interaction is the most important part related to the reciprocal O and H atoms of the Crocetin and DNA. Stabilizing interactions, obtained by ΔG calculations, showed that maximum and minimum interactions are related to the S1 and S3 regions, respectively. This means that the most probable van der Waals interaction site of the Dickerson B-DNA and Crocetin is located in the minor groove of DNA. Two sharp peaks at 2.55 and 1.75 Å in radial distribution functions of the PO?HO and NH?OC parts are related to new hydrogen bonds between the Crocetin and DNA in the complex which can be considered as the driving force of the anti-cancer mechanism of the Crocetin. Average values of 0.3 au and zero for the electron densities of the H?O bonds for DNA and complex, obtained by Quantum theory of atoms in molecules (QTAIM), means that the origin of DNA instability after complexation may be related to the H-bond denaturation by Crocetin. Finally, the evaluation of the dispersion interactions using the dispersion functional, -148.76 kcal.mol^?1, confirmed the importance of the dispersion interaction in drug-DNA complex.     

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.