Dissection, residue conservation, and structural classification of protein-DNA interfaces

The basic DNA-binding modules of 128 protein-DNA interfaces have been analyzed. Although these are less planar, like the protein-protein interfaces, the protein-DNA interfaces can also be dissected into core regions in which all the fully-buried atoms are located, and rim regions having atoms with residual accessibilities. The sequence entropy of the core residues is smaller than those in the rim, indicating that the former are better conserved and possibly contribute more towards the binding free energy, as has been implicated in protein-protein interactions. On the protein side, 1014 A(2) of the surface is buried of which 63% belong to the core. There are some differences in the propensities of residues to occur in the core and the rim. In the DNA strands, the nucleotide(s) containing fully-buried atoms in all three components usually occupy central positions of the binding region. A new classification scheme for the interfaces has been introduced based on the composition of secondary structural elements of residues and the results compared with the conventional classification of DNA-binding proteins, as well as the protein class of the molecule. It appears that a common framework may be developed to understand both protein-protein and protein-DNA interactions.     

Cavities in protein–DNA and protein–RNA interfaces

An analysis of cavities present in protein–DNA and protein–RNA complexes is presented. In terms of the number of cavities and their total volume, the interfaces formed in these complexes are akin to those in transient protein–protein heterocomplexes. With homodimeric proteins protein–DNA interfaces may contain cavities involving both the protein subunits and DNA, and these are more than twice as large as cavities involving a single protein subunit and DNA. A parameter, cavity index, measuring the degree of surface complementarity, indicates that the packing of atoms in protein–protein/DNA/RNA is very similar, but it is about two times less efficient in the permanent interfaces formed between subunits in homodimers. As within the tertiary structure and protein–protein interfaces, protein–DNA interfaces have a higher inclination to be lined by β-sheet residues; from the DNA side, base atoms, in particular those in minor grooves, have a higher tendency to be located in cavities. The larger cavities tend to be less spherical and solvated. A small fraction of water molecules are found to mediate hydrogen-bond interactions with both the components, suggesting their primary role is to fill in the void left due to the local non-complementary nature of the surface patches.     

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.     

Cavities and Atomic Packing in Protein Structures and Interfaces

A comparative analysis of cavities enclosed in a tertiary structure of proteins and interfaces formed by the interaction of two protein subunits in obligate and non-obligate categories (represented by homodimeric molecules and heterocomplexes, respectively) is presented. The total volume of cavities increases with the size of the protein (or the interface), though the exact relationship may vary in different cases. Likewise, for individual cavities also there is quantitative dependence of the volume on the number of atoms (or residues) lining the cavity. The larger cavities tend to be less spherical, solvated, and the interfaces are enriched in these. On average 15 ų of cavity volume is found to accommodate single water, with another 40–45 ų needed for each additional solvent molecule. Polar atoms/residues have a higher propensity to line solvated cavities. Relative to the frequency of occurrence in the whole structure (or interface), residues in β-strands are found more often lining the cavities, and those in turn and loop the least. Any depression in one chain not complemented by a protrusion in the other results in a cavity in the protein–protein interface. Through the use of the Voronoi volume, the packing of residues involved in protein–protein interaction has been compared to that in the protein interior. For a comparable number of atoms the interface has about twice the number of cavities relative to the tertiary structure.     

Protein-DNA interactions: A structural analysis

A detailed analysis of the DNA-binding sites of 26 proteins is presented using data from the Nucleic Acid Database (NDB) and the Protein Data Bank (PDB). Chemical and physical properties of the protein-DNA interface, such as polarity, size, shape, and packing, were analysed. The DNA-binding sites shared common features, comprising many discontinuous sequence segments forming hydrophilic surfaces capable of direct and water-mediated hydrogen bonds. These interface sites were compared to those of protein-protein binding sites, revealing them to be more polar, with many more intermolecular hydrogen bonds and buried water molecules than the protein-protein interface sites. By looking at the number and positioning of protein residue-DNA base interactions in a series of interaction footprints, three modes of DNA binding were identified (single-headed, double-headed and enveloping). Six of the eight enzymes in the data set bound in the enveloping mode, with the protein presenting a large interface area effectively wrapped around the DNA.A comparison of structural parameters of the DNA revealed that some values for the bound DNA (including twist, slide and roll) were intermediate of those observed for the unbound B-DNA and A-DNA. The distortion of bound DNA was evaluated by calculating a root-mean-square deviation on fitting to a canonical B-DNA structure. Major distortions were commonly caused by specific kinks in the DNA sequence, some resulting in the overall bending of the helix. The helix bending affected the dimensions of the grooves in the DNA, allowing the binding of protein elements that would otherwise be unable to make contact. From this structural analysis a preliminary set of rules that govern the bending of the DNA in protein-DNA complexes, are proposed.     

An analysis of the relationship between hydration and protein-DNA interactions.

Eleven protein-DNA crystal structures were analyzed to test the hypothesis that hydration sites predicted in the first hydration shell of DNA mark the positions where protein residues hydrogen-bond to DNA. For nine of those structures, protein atoms, which form hydrogen bonds to DNA bases, were found within 1.5 A of the predicted hydration positions in 86% of the interactions. The correspondence of the predicted hydration sites with the hydrogen-bonded protein side chains was significantly higher for bases inside the conserved DNA recognition sequences than outside those regions. In two CAP-DNA complexes, predicted base hydration sites correctly marked 71% (within 1.5 A) of protein atoms, which form hydrogen bonds to DNA bases. Phosphate hydration was compared to actual protein binding sites in one CAP-DNA complex with 78% marked contacts within 2.0 A. These data suggest that hydration sites mark the binding sites at protein-DNA interfaces.     

Do water molecules mediate protein-DNA recognition?

A comprehensive analysis of interfacial water molecules in the structures of 109 unique protein-DNA complexes is presented together with a new view on their role in protein-DNA recognition. Location of interfacial water molecules as reported in the crystal structures and as emerging from a series of molecular dynamics studies on protein-DNA complexes with explicit solvent and counterions, was analyzed based on their acceptor, donor hydrogen bond relationships with the atoms and residues of the macromolecules, electrostatic field calculations and packing density considerations. Water molecules for the purpose of this study have been categorized into four classes: viz. (I) those that contact both the protein and the DNA simultaneously and thus mediate recognition directly; (II) those that contact either the protein or the DNA exclusively via hydrogen bonds solvating each solute separately; (III) those that contact the hydrophobic groups in either the protein or the DNA; and, lastly (IV) those that contact another water molecule. Of the 17,963 crystallographic water molecules under examination, about 6% belong to class I and 76% belong to class II. About three-fourths of class I and class II water molecules are exclusively associated with hydrogen bond acceptor atoms of both protein and DNA. Noting that DNA is polyanionic, it is significant that a majority of the crystallographically observed water molecules as well as those from molecular dynamics simulations should be involved in facilitating binding by screening unfavorable electrostatics. Less than 2% of the reported water molecules occur between hydrogen bond donor atoms of protein and acceptor atoms of DNA. These represent cases where protein atoms cannot reach out to DNA to make favorable hydrogen bond interactions due to packing/structural restrictions and interfacial water molecules provide an extension to side-chains to accomplish hydrogen bonding.     

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.     

Display and interpretation of solvent electron density distributions in insulin crystals

In macromolecular crystallography, three-dimensional contour surfaces are useful for interactive computer graphics displays of the protein electron density but are less effective for presenting static images of large volumes of solvent density. A raster-based computer graphics program which displays depth-cued projections of continuous density distributions has been developed to analyze the distribution of solvent atoms in macromolecular crystals. Maps of the water distribution in the cubic insulin crystal show some well-ordered waters, which are bound to surrounding protein atoms by multiple hydrogen bonds, and an ill-defined solvent structure at a greater distance from the protein surface. Molecular dynamics calculations were used to assist in the interpretation of the time-varying solvent structure within two enclosed cavities in the crystal. Two water molecules that ligate a sodium ion were almost immobile during the simulation but the majority of water molecules were found to move rapidly between the density maxima identified from the crystallographic refinement.     

Contribution of cation-pi interactions to the stability of protein-DNA complexes

Cation-pi interactions between an aromatic ring and a positive charge located above it have proven to be important in protein structures and biomolecule associations. Here, the role of these interactions at the interface of protein-DNA complexes is investigated, by means of ab initio quantum mechanics energy calculations and X-ray structure analyses. Ab initio energy calculations indicate that Na ions and DNA bases can form stable cation-pi complexes, whose binding strength strongly depends on the type of base, on the position of the Na ion, and whether the base is isolated or included in a double-stranded B-DNA. A survey of protein-DNA complex structures using appropriate geometrical criteria revealed cation-pi interactions in 71% of the complexes. More than half of the cation-pi pairs involve arginine residues, about one-third asparagine or glutamine residues that only carry a partial charge, and one-seventh lysine residues. The most frequently observed pair, which is also the most stable as monitored by ab initio energy calculations, is arginine- guanine. Arginine-adenine interactions are also favorable in general, although to a lesser extent, whereas those with thymine and cytosine are not. Our calculations show that the major contribution to cation-pi interactions with DNA bases is of electrostatic nature. These interactions often occur concomitantly with hydrogen bonds with adjacent bases; their strength is estimated to be from three to four times lower than that of hydrogen bonds. Finally, the role of cation-pi interactions in the stability and specificity of protein-DNA complexes is discussed.     

Protein-protein interaction and quaternary structure

Protein-protein recognition plays an essential role in structure and function. Specific non-covalent interactions stabilize the structure of macromolecular assemblies, exemplified in this review by oligomeric proteins and the capsids of icosahedral viruses. They also allow proteins to form complexes that have a very wide range of stability and lifetimes and are involved in all cellular processes. We present some of the structure-based computational methods that have been developed to characterize the quaternary structure of oligomeric proteins and other molecular assemblies and analyze the properties of the interfaces between the subunits. We compare the size, the chemical and amino acid compositions and the atomic packing of the subunit interfaces of protein-protein complexes, oligomeric proteins, viral capsids and protein-nucleic acid complexes. These biologically significant interfaces are generally close-packed, whereas the non-specific interfaces between molecules in protein crystals are loosely packed, an observation that gives a structural basis to specific recognition. A distinction is made within each interface between a core that contains buried atoms and a solvent accessible rim. The core and the rim differ in their amino acid composition and their conservation in evolution, and the distinction helps correlating the structural data with the results of site-directed mutagenesis and in vitro studies of self-assembly.     

Dissecting subunit interfaces in homodimeric proteins

The subunit interfaces of 122 homodimers of known three-dimensional structure are analyzed and dissected into sets of surface patches by clustering atoms at the interface; 70 interfaces are single-patch, the others have up to six patches, often contributed by different structural domains. The average interface buries 1,940 A2 of the surface of each monomer, contains one or two patches burying 600-1,600 A2, is 65% nonpolar and includes 18 hydrogen bonds. However, the range of size and of hydrophobicity is wide among the 122 interfaces. Each interface has a core made of residues with atoms buried in the dimer, surrounded by a rim of residues with atoms that remain accessible to solvent. The core, which constitutes 77% of the interface on average, has an amino acid composition that resembles the protein interior except for the presence of arginine residues, whereas the rim is more like the protein surface. These properties of the interfaces in homodimers, which are permanent assemblies, are compared to those of protein-protein complexes where the components associate after they have independently folded. On average, subunit interfaces in homodimers are twice larger than in complexes, and much less polar due to the large fraction belonging to the core, although the amino acid compositions of the cores are similar in the two types of interfaces.     

Calculations of protein volumes: sensitivity analysis and parameter database

MOTIVATION: The precise sizes of protein atoms in terms of occupied packing volume are of great importance. We have previously presented standard volumes for protein residues based on calculations with Voronoi-like polyhedra. To understand the applicability and limitations of our set, we investigated, in detail, the sensitivity of the volume calculations to a number of factors: (i) the van der Waals radii set, (ii) the criteria for including buried atoms in the calculations or atom selection, (iii) the method of positioning the dividing plane in polyhedra construction, and (iv) the set of structures used in the averaging. RESULTS: We find that different radii sets have only moderate affects to the distribution and mean of volumes. Atom selection and dividing plane methods cause larger changes in protein atoms volumes. More significantly, we show how the variation in volumes appears to be clearly related to the quality of the structures analyzed, with higher quality structures giving consistently smaller average volumes with less variance.     

Visualizing the Behavior of Human Rad51 at the Single-Molecule Level

The repair of double-stranded DNA breaks by homologous recombination is essential for maintaining genome integrity. Much of what we know about this DNA repair pathway in eukaryotes has been gleaned from genetics, in vivo experiments with GFP-tagged proteins and traditional biochemical experiments with purified proteins. However, many questions have remained inaccessible to these experimental approaches. Recent technological advances have made it possible to directly visualize the behaviors of individual DNA and protein molecules in vitro, and it is now becoming feasible to apply these technology-driven approaches to complex biochemical systems, such as those involved in the repair of damaged DNA. This report summarizes the use of total internal reflection fluorescence microscopy to probe fundamental aspects of protein-DNA interactions at the single-molecule level, and specific emphasis is placed on our efforts to develop new methods and techniques for studying DNA repair. Using these new approaches we are investigating the DNA-binding behavior of human Rad51 and we have recently demonstrated that this protein can slide on dsDNA via a one-dimensional random walk mechanism driven solely by thermal fluctuations of the surrounding solvent. Here, we highlight some possible implications of this recent finding, and we also briefly discuss the potential benefits of future single-molecule studies in the study of protein-DNA interactions and DNA repair.     

Cavities and packing at protein interfaces.

An analysis of internal packing defects or "cavities" (both empty and water-containing) within protein structures has been undertaken and includes 3 cavity classes: within domains, between domains, and between protein subunits. We confirm several basic features common to all cavity types but also find a number of new characteristics, including those that distinguish the classes. The total cavity volume remains only a small fraction of the total protein volume and yet increases with protein size. Water-filled "cavities" possess a more polar surface and are typically larger. Their constituent waters are necessary to satisfy the local hydrogen bonding potential. Cavity-surrounding atoms are observed to be, on average, less flexible than their environments. Intersubunit and interdomain cavities are on average larger than the intradomain cavities, occupy a larger fraction of their resident surfaces, and are more frequently water-filled. We observe increased cavity volume at domain-domain interfaces involved with shear type domain motions. The significance of interfacial cavities upon subunit and domain shape complementarity and the protein docking problem, as well as in their structural and functional role in oligomeric proteins, will be discussed. The results concerning cavity size, polarity, solvation, general abundance, and residue type constituency should provide useful guidelines for protein modeling and design.