Structural segments and residue propensities in protein-RNA interfaces: comparison with protein-protein and protein-DNA complexes

The interface of a protein molecule that is involved in binding another protein, DNA or RNA has been characterized in terms of the number of unique secondary structural segments (SSSs), made up of stretches of helix, strand and non-regular (NR) regions. On average 10-11 segments define the protein interface in protein-protein (PP) and protein-DNA (PD) complexes, while the number is higher (14) for protein-RNA (PR) complexes. While the length of helical segments in PP interaction increases with the interface area, this is not the case in PD and PR complexes. The propensities of residues to occur in the three types of secondary structural elements (SSEs) in the interface relative to the corresponding elements in the protein tertiary structures have been calculated. Arg, Lys, Asn, Tyr, His and Gln are preferred residues in PR complexes; in addition, Ser and Thr are also favoured in PD interfaces.     

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.     

Dissecting protein-RNA recognition sites

We analyze the protein–RNA interfaces in 81 transient binary complexes taken from the Protein Data Bank. Those with tRNA or duplex RNA are larger than with single-stranded RNA, and comparable in size to protein–DNA interfaces. The protein side bears a strong positive electrostatic potential and resembles protein–DNA interfaces in its amino acid composition. On the RNA side, the phosphate contributes less, and the sugar much more, to the interaction than in protein–DNA complexes. On average, protein–RNA interfaces contain 20 hydrogen bonds, 7 that involve the phosphates, 5 the sugar 2′OH, and 6 the bases, and 32 water molecules. The average H-bond density per unit buried surface area is less with tRNA or single-stranded RNA than with duplex RNA. The atomic packing is also less compact in interfaces with tRNA. On the protein side, the main chain NH and Arg/Lys side chains account for nearly half of all H-bonds to RNA; the main chain CO and side chain acceptor groups, for a quarter. The 2′OH is a major player in protein–RNA recognition, and shape complementarity an important determinant, whereas electrostatics and direct base–protein interactions play a lesser part than in protein–DNA recognition.     

Dissecting water binding sites at protein–protein interfaces: a lesson from the atomic structures in the Protein Data Bank

We dissect the protein–protein interfaces into water preservation (WP), water hydration (WH) and water dehydration (WD) sites by comparing the water-mediated hydrogen bonds (H-bond) in the bound and unbound states of the interacting subunits. Upon subunit complexation, if a H-bond between an interface water and a protein polar group is retained, we assign it as WP site; if it is lost, we assign it as WD site and if a new H-bond is created, we assign it as WH site. We find that the density of WD sites is highest followed by WH and WP sites except in antigen and (or) antibody complexes, where the density of WH sites is highest followed by WD and WP sites. Furthermore, we find that WP sites are the most conserved followed by WD and WH sites in all class of complexes except in antigen and (or) antibody complexes, where WD sites are the most conserved followed by WH and WP sites. A significant number of WP and WH sites are involved in water bridges that stabilize the subunit interactions. At WH sites, the residues involved in water bridges are significantly better conserved than the other residues. However, no such difference is observed at WP sites. Interestingly, WD sites are generally replaced with direct H-bonds upon subunit complexation. Significantly, we observe many water-mediated H-bonds remain preserved in spite of large conformational changes upon subunit complexation. These findings have implications in predicting and engineering water binding sites at protein–protein interfaces.     

Dissecting the protein-RNA interface: the role of protein surface shapes and RNA secondary structures in protein-RNA recognition

Protein-RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein-RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein-RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating 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.     

Partner-specific prediction of RNA-binding residues in proteins: A critical assessment

RNA-protein interactions play essential roles in regulating gene expression. While some RNA-protein interactions are “specific”, that is, the RNA-binding proteins preferentially bind to particular RNA sequence or structural motifs, others are “non-RNA specific.” Deciphering the protein-RNA recognition code is essential for comprehending the functional implications of these interactions and for developing new therapies for many diseases. Because of the high cost of experimental determination of protein-RNA interfaces, there is a need for computational methods to identify RNA-binding residues in proteins. While most of the existing computational methods for predicting RNA-binding residues in RNA-binding proteins are oblivious to the characteristics of the partner RNA, there is growing interest in methods for partner-specific prediction of RNA binding sites in proteins. In this work, we assess the performance of two recently published partner-specific protein-RNA interface prediction tools, PS-PRIP, and PRIdictor, along with our own new tools. Specifically, we introduce a novel metric, RNA-specificity metric (RSM), for quantifying the RNA-specificity of the RNA binding residues predicted by such tools. Our results show that the RNA-binding residues predicted by previously published methods are oblivious to the characteristics of the putative RNA binding partner. Moreover, when evaluated using partner-agnostic metrics, RNA partner-specific methods are outperformed by the state-of-the-art partner-agnostic methods. We conjecture that either (a) the protein-RNA complexes in PDB are not representative of the protein-RNA interactions in nature, or (b) the current methods for partner-specific prediction of RNA-binding residues in proteins fail to account for the differences in RNA partner-specific versus partner-agnostic protein-RNA interactions, or both.     

Hydration of protein–RNA recognition sites

We investigate the role of water molecules in 89 protein–RNA complexes taken from the Protein Data Bank. Those with tRNA and single-stranded RNA are less hydrated than with duplex or ribosomal proteins. Protein–RNA interfaces are hydrated less than protein–DNA interfaces, but more than protein–protein interfaces. Majority of the waters at protein–RNA interfaces makes multiple H-bonds; however, a fraction do not make any. Those making H-bonds have preferences for the polar groups of RNA than its partner protein. The spatial distribution of waters makes interfaces with ribosomal proteins and single-stranded RNA relatively ‘dry’ than interfaces with tRNA and duplex RNA. In contrast to protein–DNA interfaces, mainly due to the presence of the 2′OH, the ribose in protein–RNA interfaces is hydrated more than the phosphate or the bases. The minor groove in protein–RNA interfaces is hydrated more than the major groove, while in protein–DNA interfaces it is reverse. The strands make the highest number of water-mediated H-bonds per unit interface area followed by the helices and the non-regular structures. The preserved waters at protein–RNA interfaces make higher number of H-bonds than the other waters. Preserved waters contribute toward the affinity in protein–RNA recognition and should be carefully treated while engineering protein–RNA interfaces.     

Standard atomic volumes in double-stranded DNA and packing in protein--DNA interfaces

Standard volumes for atoms in double-stranded B-DNA are derived using high resolution crystal structures from the Nucleic Acid Database (NDB) and compared with corresponding values derived from crystal structures of small organic compounds in the Cambridge Structural Database (CSD). Two different methods are used to compute these volumes: the classical Voronoi method, which does not depend on the size of atoms, and the related Radical Planes method which does. Results show that atomic groups buried in the interior of double-stranded DNA are, on average, more tightly packed than in related small molecules in the CSD. The packing efficiency of DNA atoms at the interfaces of 25 high resolution protein-DNA complexes is determined by computing the ratios between the volumes of interfacial DNA atoms and the corresponding standard volumes. These ratios are found to be close to unity, indicating that the DNA atoms at protein-DNA interfaces are as closely packed as in crystals of B-DNA. Analogous volume ratios, computed for buried protein atoms, are also near unity, confirming our earlier conclusions that the packing efficiency of these atoms is similar to that in the protein interior. In addition, we examine the number, volume and solvent occupation of cavities located at the protein-DNA interfaces and compared them with those in the protein interior. Cavities are found to be ubiquitous in the interfaces as well as inside the protein moieties. The frequency of solvent occupation of cavities is however higher in the interfaces, indicating that those are more hydrated than protein interiors. Lastly, we compare our results with those obtained using two different measures of shape complementarity of the analysed interfaces, and find that the correlation between our volume ratios and these measures, as well as between the measures themselves, is weak. Our results indicate that a tightly packed environment made up of DNA, protein and solvent atoms plays a significant role in protein-DNA recognition.     

A Comparison of Structural and Evolutionary Attributes of Escherichia coli and Thermus thermophilus Small Ribosomal Subunits: Signatures of Thermal Adaptation

Here we compare the structural and evolutionary attributes of Thermus thermophilus and Escherichia coli small ribosomal subunits (SSU). Our results indicate that with few exceptions, thermophilic 16S ribosomal RNA (16S rRNA) is densely packed compared to that of mesophilic at most of the analogous spatial regions. In addition, we have located species-specific cavity clusters (SSCCs) in both species. E. coli SSCCs are numerous and larger compared to T. thermophilus SSCCs, which again indicates densely packed thermophilic 16S rRNA. Thermophilic ribosomal proteins (r-proteins) have longer disordered regions than their mesophilic homologs and they experience larger disorder-to-order transitions during SSU-assembly. This is reflected in the predicted higher conformational changes of thermophilic r-proteins compared to their mesophilic homologs during SSU-assembly. This high conformational change of thermophilic r-proteins may help them to associate with the 16S ribosomal RNA with high complementary interfaces, larger interface areas, and denser molecular contacts, compared to those of mesophilic. Thus, thermophilic protein-rRNA interfaces are tightly associated with 16S rRNA than their mesophilic homologs. Densely packed 16S rRNA interior and tight protein-rRNA binding of T. thermophilus (compared to those of E. coli) are likely the signatures of its thermal adaptation. We have found a linear correlation between the free energy of protein-RNA interface formation, interface size, and square of conformational changes, which is followed in both prokaryotic and eukaryotic SSU. Disorder is associated with high protein-RNA interface polarity. We have found an evolutionary tendency to maintain high polarity (thereby disorder) at protein-rRNA interfaces, than that at rest of the protein structures. However, some proteins exhibit exceptions to this general trend.     

A protein-RNA docking benchmark (I): nonredundant cases

We have developed a nonredundant protein-RNA docking benchmark dataset, which is derived from the available bound and unbound structures in the Protein Data Bank involving polypeptide and nucleic acid chains. It consists of nine unbound-unbound cases where both the protein and the RNA are available in the free form. The other 36 cases are of unbound-bound type where only the protein is available in the free form. The conformational change upon complex formation is calculated by a distance matrix alignment method, and based on that, complexes are classified into rigid, semi-flexible, and full flexible. Although in the rigid body category, no significant conformational change accompanies complex formation, the fully flexible test cases show large domain movements, RNA base flips, etc. The benchmark covers four major groups of RNA, namely, t-RNA, ribosomal RNA, duplex RNA, and single-stranded RNA. We find that RNA is generally more flexible than the protein in the complexes, and the interface region is as flexible as the molecule as a whole. The structural diversity of the complexes in the benchmark set should provide a common ground for the development and comparison of the protein-RNA docking methods. The benchmark can be freely downloaded from the internet.     

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA

We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure. Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA. Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later. We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit.     

Charged residues at protein interaction interfaces: unexpected conservation and orchestrated divergence

Protein-protein interactions play an essential role in the functioning of cell. The importance of charged residues and their diverse role in protein-protein interactions have been well studied using experimental and computational methods. Often, charged residues located in protein interaction interfaces are conserved across the families of homologous proteins and protein complexes. However, on a large scale, it has been recently shown that charged residues are significantly less conserved than other residue types in protein interaction interfaces. The goal of this work is to understand the role of charged residues in the protein interaction interfaces through their conservation patterns. Here, we propose a simple approach where the structural conservation of the charged residue pairs is analyzed among the pairs of homologous binary complexes. Specifically, we determine a large set of homologous interactions using an interaction interface similarity measure and catalog the basic types of conservation patterns among the charged residue pairs. We find an unexpected conservation pattern, which we call the correlated reappearance, occurring among the pairs of homologous interfaces more frequently than the fully conserved pairs of charged residues. Furthermore, the analysis of the conservation patterns across different superkingdoms as well as structural classes of proteins has revealed that the correlated reappearance of charged residues is by far the most prevalent conservation pattern, often occurring more frequently than the unconserved charged residues. We discuss a possible role that the new conservation pattern may play in the long-range electrostatic steering effect.     

The Subunit Interfaces of Weakly Associated Homodimeric Proteins

We analyzed subunit interfaces in 315 homodimers with an X-ray structure in the Protein Data Bank, validated by checking the literature for data that indicate that the proteins are dimeric in solution and that, in the case of the “weak” dimers, the homodimer is in equilibrium with the monomer. The interfaces of the 42 weak dimers, which are smaller by a factor of 2.4 on average than in the remainder of the set, are comparable in size with antibody-antigen or protease-inhibitor interfaces. Nevertheless, they are more hydrophobic than in the average transient protein-protein complex and similar in amino acid composition to the other homodimer interfaces. The mean numbers of interface hydrogen bonds and hydration water molecules per unit area are also similar in homodimers and transient complexes. Parameters related to the atomic packing suggest that many of the weak dimer interfaces are loosely packed, and we suggest that this contributes to their low stability. To evaluate the evolutionary selection pressure on interface residues, we calculated the Shannon entropy of homologous amino acid sequences at 60% sequence identity. In 93% of the homodimers, the interface residues are better conserved than the residues on the protein surface. The weak dimers display the same high degree of interface conservation as other homodimers, but their homologs may be heterodimers as well as homodimers. Their interfaces may be good models in terms of their size, composition, and evolutionary conservation for the labile subunit contacts that allow protein assemblies to share and exchange components, allosteric proteins to undergo quaternary structure transitions, and molecular machines to operate in the cell.     

蛋白质-核酸复合物界面氨基酸与核苷酸偏好性分析

蛋白质-核酸相互作用机制到目前还不是很清楚,尤其是蛋白质与RNA的相互作用。目前,可得到的蛋白质-核酸复合物结构数据不断增多,作者收集了Protein Data Bank数据库中所有的蛋白质-核酸复合物结构数据,对复合物中结合残基和结合核苷酸的偏好性进行了统计分析。发现：1）不同功能的蛋白质-核酸复合物间的结合残基数量存在显著差异;2）在蛋白 质-DNA和蛋白质-RNA复合物界面,碱性氨基酸都是最受欢迎的;3）氨基酸的极性大小及方向在决定它是否与RNA分子进行结合时起到重要的作用,同时发现氨基酸侧链形成的空间位阻会影响氨基酸残基与RNA分子的相互作用;4）随着定义结合残基距离阈值的增大,其氨基酸使用的特异性降低,而受欢迎与不受欢迎的氨基酸种类均没有变化。     

A dissection of the protein-protein interfaces in icosahedral virus capsids

We selected 49 icosahedral virus capsids whose crystal structures are reported in the Protein Data Bank. They belong to the T=1, T=3, pseudo T=3 and other lattice types. We identified in them 779 unique interfaces between pairs of subunits, all repeated by icosahedral symmetry. We analyzed the geometric and physical chemical properties of these interfaces and compared with interfaces in protein-protein complexes and homodimeric proteins, and with crystal packing contacts. The capsids contain one to 16 subunits implicated in three to 66 unique interfaces. Each subunit loses 40-60% of its accessible surface in contacts with an average of 8.5 neighbors. Many of the interfaces are very large with a buried surface area (BSA) that can exceed 10,000 A(2), yet 39% are small with a BSA<800 A(2) comparable to crystal packing contacts. Pairwise capsid interfaces overlap, so that one-third of the residues are part of more than one interface. Those with a BSA>800 A(2) resemble homodimer interfaces in their chemical composition. Relative to the protein surface, they are non-polar, enriched in aliphatic residues and depleted of charged residues, but not of neutral polar residues. They contain one H-bond per about 200 A(2) BSA. Small capsid interfaces (BSA<800 A(2)) are only slightly more polar. They have a similar amino acid composition, but they bury fewer atoms and contain fewer H-bonds for their size. Geometric parameters that estimate the quality of the atomic packing suggest that the small capsid interfaces are loosely packed like crystal packing contacts, whereas the larger interfaces are close-packed as in protein-protein complexes and homodimers. We discuss implications of these findings on the mechanism of capsid assembly, assuming that the larger interfaces form first to yield stable oligomeric species (capsomeres), and that medium-size interfaces allow the stepwise addition of capsomeres to build larger intermediates.