Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions

We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid–ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine.     

On the molecular discrimination between adenine and guanine by proteins

The molecular recognition and discrimination of adenine and guanine ligand moieties in complexes with proteins have been studied using empirical observations on carefully selected crystal structures. The distribution of protein folds that bind these purines has been found to differ significantly from that across the whole PDB, but the most populated architectures and folds are also the most common in three genomes from the three different domains of life. The protein environments around the two nucleic acid bases were significantly different, in terms of the propensities of amino acid residues to be in the binding site, as well as their propensities to form hydrogen bonds to the bases. Plots of the distribution of protein atoms around the two purines clearly show different clustering of hydrogen bond donors and acceptors opposite complimentary acceptors and donors in the rings, with hydrophobic areas below and above the rings. However, the clustering pattern is fuzzy, reflecting the variety of ways that proteins have evolved to recognise the same molecular moiety. Furthermore, an analysis of the conservation of residues in the protein chains binding guanine shows that residues in contact with the base are in general better conserved than the rest of the chain.     

Specific interactions for ab initio folding of protein terminal regions with secondary structures

Proteins fold into unique three-dimensional structures by specific, orientation-dependent interactions between amino acid residues. Here, we extract orientation-dependent interactions from protein structures by treating each polar atom as a dipole with a direction. The resulting statistical energy function successfully refolds 13 out of 16 fully unfolded secondary-structure terminal regions of 10-23 amino acid residues in 15 small proteins. Dissecting the orientation-dependent energy function reveals that the orientation preference between hydrogen-bonded atoms is not enough to account for the structural specificity of proteins. The result has significant implications on the theoretical and experimental searches for specific interactions involved in protein folding and molecular recognition between proteins and other biologically active molecules.     

The role of RNA sequence and structure in RNA--protein interactions

We investigate the sequence and structural properties of RNA-protein interaction sites in 211 RNA-protein chain pairs, the largest set of RNA-protein complexes analyzed to date. Statistical analysis confirms and extends earlier analyses made on smaller data sets. There are 24.6% of hydrogen bonds between RNA and protein that are nucleobase specific, indicating the importance of both nucleobase-specific and -nonspecific interactions. While there is no significant difference between RNA base frequencies in protein-binding and non-binding regions, distinct preferences for RNA bases, RNA structural states, protein residues, and protein secondary structure emerge when nucleobase-specific and -nonspecific interactions are considered separately. Guanine nucleobase and unpaired RNA structural states are significantly preferred in nucleobase-specific interactions; however, nonspecific interactions disfavor guanine, while still favoring unpaired RNA structural states. The opposite preferences of nucleobase-specific and -nonspecific interactions for guanine may explain discrepancies between earlier studies with regard to base preferences in RNA-protein interaction regions. Preferences for amino acid residues differ significantly between nucleobase-specific and -nonspecific interactions, with nonspecific interactions showing the expected bias towards positively charged residues. Irregular protein structures are strongly favored in interactions with the protein backbone, whereas there is little preference for specific protein secondary structure in either nucleobase-specific interaction or -nonspecific interaction. Overall, this study shows strong preferences for both RNA bases and RNA structural states in protein-RNA interactions, indicating their mutual importance in protein recognition.     

Non-hydrogen bond interactions involving the methionine sulfur atom

Of all the nonbonded interactions, hydrogen bond, because of its geometry involving polar atoms, is the most easily recognizable. Here we characterize two interactions involving the divalent sulfur of methionine (Met) residues that do not need any participation of proton. In one an oxygen atom of the main-chain carbonyl group or a carboxylate side chain is used. In another an aromatic atom interacting along the face of the ring is utilized. In these, the divalent sulfur behaves as an electrophile and the other electron-rich atom, a nucleophile. The stereochemistry of the interaction is such that the nucleophile tends to approach approximately along the extension of one of the covalent bonds to S. The nitrogen atom of histidine side chain is extensively used in these nonbonded contacts. There is no particular geometric pattern in the interaction of S with the edge of an aromatic ring, except when an N-H group in involved, which is found within 40 degrees from the perpendicular to the sulfide plane, thus defining the geometry of hydrogen bond interaction involving the sulfur atom. As most of the Met residues which partake in such stereospecific interactions are buried, these would be important for the stability of the protein core, and their incorporation in the binding site would be useful for molecular recognition and optimization of the site's affinity for partners (especially containing aromatic and heteroaromatic groups). Mutational studies aimed at replacing Met by other residues would benefit from the delineation of these interactions.     

Analysis of the structural determinants for RNA binding of the human protein AUF1/hnRNP D

The protein AUF1/hnRNP D was one of the first factors identified that binds to the AU-rich region of certain mRNAs and mediates their fast degradation. Here we describe experiments to address the structural determinants for the binding of AUF1 to the RNA by combining comparative molecular modeling with gel shift assays. From our model of the RNA binding region of AUF1 we predicted that it interacts with RNA predominantly through stacking interactions that do not provide base-specific recognition. Only two RNA positions bound by AUF1 show base preferences: one for pyrimidine bases and the second for a conserved adenine residue. Gel shift assays with a panel of RNA oligonucleotides largely confirmed these model-based binding determinants. An alignment with proteins of the hnRNP family demonstrated that the amino acids involved in the stacking interactions are conserved whereas those that confer a base-specific recognition in AUF1 are variable.     

Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins

Amino acids in peptides and proteins display distinct preferences for alpha-helical, beta-strand, and other conformational states. Various physicochemical reasons for these preferences have been suggested: conformational entropy, steric factors, hydrophobic effect, and backbone electrostatics; however, the issue remains controversial. It has been proposed recently that the side-chain-dependent solvent screening of the local and non-local backbone electrostatic interactions primarily determines the preferences not only for the alpha-helical but also for all other main-chain conformational states. Side-chains modulate the electrostatic screening of backbone interactions by excluding the solvent from the vicinity of main-chain polar atoms. The deficiency of this electrostatic screening model of amino acid preferences is that the relationships between the main-chain electrostatics and the amino acid preferences have been demonstrated for a limited set of six non-polar amino acid types in proteins only. Here, these relationships are determined for all amino acid types in tripeptides, dekapeptides, and proteins. The solvation free energies of polar backbone atoms are approximated by the electrostatic contributions calculated by the finite difference Poisson-Boltzmann and the Langevin dipoles methods. The results show that the average solvation free energy of main-chain polar atoms depends strongly on backbone conformation, shape of side-chains, and exposure to solvent. The equilibrium between the low-energy beta-strand conformation of an amino acid (anti-parallel alignment of backbone dipole moments) and the high-energy alpha conformation (parallel alignment of backbone dipole moments) is strongly influenced by the solvation of backbone polar atoms. The free energy cost of reaching the alpha conformation is by approximately 1.5 kcal/mol smaller for residues with short side-chains than it is for the large beta-branched amino acid residues. This free energy difference is comparable to those obtained experimentally by mutation studies and is thus large enough to account for the distinct preferences of amino acid residues. The screening coefficients gamma(local)(r) and gamma(non-local)(r) correlate with the solvation effects for 19 amino acid types with the coefficients between 0.698 to 0.851, depending on the type of calculation and on the set of point atomic charges used. The screening coefficients gamma(local)(r) increase with the level of burial of amino acids in proteins, converging to 1.0 for the completely buried amino acid residues. The backbone solvation free energies of amino acid residues involved in strong hydrogen bonding (for example: in the middle of an alpha-helix) are small. The hydrogen bonded backbone is thus more hydrophobic than the peptide groups in random coil. The alpha-helix forming preference of alanine is attributed to the relatively small free energy cost of reaching the high-energy alpha-helix conformation. These results confirm that the side-chain-dependent solvent screening of the backbone electrostatic interactions is the dominant factor in determining amino acid conformational preferences.     

Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity

We investigate the conservation of amino acid residue sequences in 21 DNA-binding protein families and study the effects that mutations have on DNA-sequence recognition. The observations are best understood by assigning each protein family to one of three classes: (i) non-specific, where binding is independent of DNA sequence; (ii) highly specific, where binding is specific and all members of the family target the same DNA sequence; and (iii) multi-specific, where binding is also specific, but individual family members target different DNA sequences. Overall, protein residues in contact with the DNA are better conserved than the rest of the protein surface, but there is a complex underlying trend of conservation for individual residue positions. Amino acid residues that interact with the DNA backbone are well conserved across all protein families and provide a core of stabilising contacts for homologous protein-DNA complexes. In contrast, amino acid residues that interact with DNA bases have variable levels of conservation depending on the family classification. In non-specific families, base-contacting residues are well conserved and interactions are always found in the minor groove where there is little discrimination between base types. In highly specific families, base-contacting residues are highly conserved and allow member proteins to recognise the same target sequence. In multi-specific families, base-contacting residues undergo frequent mutations and enable different proteins to recognise distinct target sequences. Finally, we report that interactions with bases in the target sequence often follow (though not always) a universal code of amino acid-base recognition and the effects of amino acid mutations can be most easily understood for these interactions.     

Interaction of DNA with clusters of amino acids in proteins

Protein-DNA interactions facilitate the fundamental functions of living cells and are universal in all living organisms. Several investigations have been carried out, essentially identifying pairs of interactions between the amino acid residues in proteins and the bases in DNA. In the present study, we have detected the recognition motifs that may constitute a cluster of spatially interacting residues in proteins, which interact with the bases of DNA. Graph spectral algorithm has been used to detect side chain clusters comprising Arg, Lys, Asn, Gln and aromatic residues from proteins interacting with DNA. We find that the interaction of proteins with DNA is through clusters in about half of the proteins in the dataset and through individual residues in the rest. Furthermore, inspection of the clusters has revealed additional interactions in a few cases, which have not been reported earlier. The geometry of the interaction between the DNA base and the protein residue is quantified by the distance d and the angle theta. These parameters have been identified for the cation-pi/H-bond stair motif that was reported earlier. Among the Arg, Lys, Asn and Gln residues, the range of (d, theta) values of the interacting Arg clearly falls into the cation-pi and the hydrogen bond interactions of the 'cation-pi/H-bond' stair motif. Analysis of the cluster composition reveals that the Arg residue is predominant than the Lys, Asn and Gln residues. The clusters are classified into Type I and Type II based on the presence or absence of aromatic residues (Phe, Tyr) in them. Residue conservation in these clusters has been examined. Apart from the conserved residues identified previously, a few more residues mainly Phe, Tyr and Arg have also been identified as conserved and interactive with the DNA. Interestingly, a few residues that are parts of interacting clusters and do not interact directly with the DNA have also been conserved. This emphasizes the importance of recognizing the protein side chain cluster motifs interacting with the DNA, which could serve as signatures of protein-DNA recognition in the families of DNA binding proteins.     

Insights into DNA solvation found in protein-DNA structures

The proteins that bind double-helical DNA present various microenvironments that sense and/or induce signals in the genetic material. The high-resolution structures of protein-DNA complexes reveal the nature of both the microenvironments and the conformational responses in DNA and protein. Complex networks of interactions within the structures somehow tie the protein and DNA together and induce the observed spatial forms. Here we show how the cumulative buildup of amino acid atoms around the sugars, phosphates, and bases in different protein-DNA complexes produces a binding cloud around the double helix and how different types of atoms fill that cloud. Rather than focusing on the principles of molecular binding and recognition suggested by the arrangements of amino acids and nucleotides in the macromolecular complexes, we consider the proteins in contact with DNA as organized solvents. We describe differences in the mix of atoms that come in closest contact with DNA, subtle sequence-dependent features in the microenvironment of the sugar-phosphate backbone, a direct link between the localized buildup of ionic species and the electrostatic potential surfaces of the DNA bases, and sites of atomic buildup above and below the basepair planes that transmit the unique features of the base environments along the chain backbone. The inferences about solvation that can be drawn from the survey provide new stimuli for improvement of nucleic acid force fields and fresh ideas for exploration of the properties of DNA in solution.     

A method to search for similar protein local structures at ligand-binding sites and its application to adenine recognition

We have developed a method of searching for similar spatial arrangements of atoms around a given chemical moiety in proteins that bind a common ligand. The first step in this method is to consider a set of atoms that closely surround a given chemical moiety. Then, to compare the spatial arrangements of such surrounding atoms in different proteins, they are translated and rotated so that the chemical moieties are superposed on each other. Spatial arrangements of surrounding atoms in a pair of proteins are judged to be similar, when there are many corresponding atoms occupying similar spatial positions. Because the method focuses on the arrangements of surrounding atoms, it can detect structural similarities of binding sites in proteins that are dissimilar in their amino acid sequences or in their chain folds. We have applied this method to identify modes of nucleotide base recognition by proteins. An all-against-all comparison of the arrangements of atoms surrounding adenine moieties revealed an unexpected structural similarity between protein kinases, cAMP-dependent protein kinase (cAPK), and casein kinase-1 (CK1), and D-Ala:D-Ala ligase (DD-ligase) at their adenine-binding sites, despite a lack of similarity in their chain folds. The similar local structure consists of a four-residue segment and three sequentially separated residues. In particular the four-residue segments of these enzymes were found to have nearly identical conformations in their backbone parts, which are involved in the recognition of adenine. This common local structure was also found in substrate-free three-dimensional structures of other proteins that are similar to DD-ligase in the chain fold and of other protein kinases. As the proteins with different folds were found to share a common local structure, these proteins seem to constitute a remarkable example of convergent evolution for the same recognition mechanism. Received: 9 December 1996 / Accepted: 7 February 1997     

Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.     

Structural mechanisms of the degenerate sequence recognition by Bse634I restriction endonuclease

Restriction endonuclease Bse634I recognizes and cleaves the degenerate DNA sequence 5'-R/CCGGY-3' (R stands for A or G; Y for T or C, '/' indicates a cleavage position). Here, we report the crystal structures of the Bse634I R226A mutant complexed with cognate oligoduplexes containing ACCGGT and GCCGGC sites, respectively. In the crystal, all potential H-bond donor and acceptor atoms on the base edges of the conserved CCGG core are engaged in the interactions with Bse634I amino acid residues located on the α6 helix. In contrast, direct contacts between the protein and outer base pairs are limited to van der Waals contact between the purine nucleobase and Pro203 residue in the major groove and a single H-bond between the O2 atom of the outer pyrimidine and the side chain of the Asn73 residue in the minor groove. Structural data coupled with biochemical experiments suggest that both van der Waals interactions and indirect readout contribute to the discrimination of the degenerate base pair by Bse634I. Structure comparison between related enzymes Bse634I (R/CCGGY), NgoMIV (G/CCGGC) and SgrAI (CR/CCGGYG) reveals how different specificities are achieved within a conserved structural core.     

Stacking interactions in PUF-RNA complexes

Stacking interactions between amino acids and bases are common in RNA-protein interactions. Many proteins that regulate mRNAs interact with single-stranded RNA elements in the 3' UTR (3'-untranslated region) of their targets. PUF proteins are exemplary. Here we focus on complexes formed between a Caenorhabditis elegans PUF protein, FBF, and its cognate RNAs. Stacking interactions are particularly prominent and involve every RNA base in the recognition element. To assess the contribution of stacking interactions to formation of the RNA-protein complex, we combine in vivo selection experiments with site-directed mutagenesis, biochemistry, and structural analysis. Our results reveal that the identities of stacking amino acids in FBF affect both the affinity and specificity of the RNA-protein interaction. Substitutions in amino acid side chains can restrict or broaden RNA specificity. We conclude that the identities of stacking residues are important in achieving the natural specificities of PUF proteins. Similarly, in PUF proteins engineered to bind new RNA sequences, the identity of stacking residues may contribute to "target" versus "off-target" interactions, and thus be an important consideration in the design of proteins with new specificities.     

The three-dimensional structure of bovine calcium ion-bound osteocalcin using 1H NMR spectroscopy

Structural information on osteocalcin or other noncollagenous bone proteins is very limited. We have solved the three-dimensional structure of calcium bound osteocalcin using (1)H 2D NMR techniques and proposed a mechanism for mineral binding. The protons in the 49 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. Distance constraints, dihedral angle constraints, hydrogen bonds, and (1)H and (13)C chemical shifts were all used to calculate a family of 13 structures. The tertiary structure of the protein consisted of an unstructured N terminus and a C-terminal loop (residues 16-49) formed by long-range hydrophobic interactions. Elements of secondary structure within residues 16-49 include type III turns (residues 20-25) and two alpha-helical regions (residues 27-35 and 41-44). The three Gla residues project from the same face of the helical turns and are surface exposed. The genetic algorithm-molecular dynamics simulation approach was used to place three calcium atoms on the NMR-derived structure. One calcium atom was coordinated by three side chain oxygen atoms, two from Asp30, and one from Gla24. The second calcium atom was coordinated to four oxygen atoms, two from the side chain in Gla 24, and two from the side chain of Gla 21. The third calcium atom was coordinated to two oxygen atoms of the side chain of Gla17. The best correlation of the distances between the uncoordinated Gla oxygen atoms is with the intercalcium distance of 9.43 A in hydroxyapatite. The structure may provide further insight into the function of osteocalcin.     

Thymine methyls and DNA-protein interactions.

Evidence is summarized showing that thymine methyls are as important in the recognition of specific sequences by proteins as are the more widely recognized hydrogen bonding sites of bases in the major groove (1). Strongest evidence has come from experiments using functional group mutagenesis (2) in which thymines in a specific recognition sequence (e.g., promoters, operators and restriction sites) are replaced by oligonucleotide synthesis with methyl-free uracil or cytosine and 5-methylcytosine. Such experiments have shown that thymine methyls can provide contact points via van der Waals interactions with amino acid side chains of specific DNA binding proteins. Actual contact between a thymine methyl and carbons of a glutamine side chain has been observed in a cocrystal of the phage 434 repressor and its operator by X-ray analysis. The issue of why thymine occurs in DNA is discussed in light of these findings.     

Purification and characterization of cytochrome c3, ferredoxin, and rubredoxin isolated from Desulfovibrio desulfuricans Norway.

Different electron carriers of the non-desulfoviridin-containing, sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) have been studied. Two nonheme iron proteins, ferredoxin and rubredoxin, have been purified. This ferredoxin contains four atoms of non-heme iron and acid-labile sulfur and six residues of cysteine per molecule. Its amino acid composition suggests that it is homologous with the other Desulfovibrio ferredoxins. The rubredoxin is also an acidic protein of 6,000 molecular weight and contains one atom of iron and four cysteine residues per molecule. The amino acid composition and molecular weight of the cytochrome c3 from D. desulfuricans (strain Norway 4) are reported. Its spectral properties are very similar to those of the other cytochromes c3 (molecular weight, 13,000) of Desulfovibrio and show that it contains four hemes per molecule. This cytochrome has a very low redox potential and acts as a carrier in the coupling of hydrogenase and thiosulfate reductase in extracts of Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain) in contrast to D. gigas cytochrome c3 (molecular weight, 13,000). A comparison of the activities of the cytochrome c3 (molecular weight, 13,000) of D. gigas and that of D. desulfuricans in this reaction suggests that these homologous proteins can have different specificity in the electron transfer chain of these bacteria.     

Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket

mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.     

2-Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5' em leader CC*T/AGG em leader 3' DNA sequence and catalyzes the transfer of the methyl group from S-adenosyl-l-methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2-pyrimidinone, a cytosine analogue lacking an NH(2) group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5' em leader CCT/AGG em leader 3' sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2-pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2-pyrimidinone and to a greater destabilizing influence of 2-pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2-pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA-M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.EcoRII. In the absence of the cofactor S-adenosyl-l-methionine, proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of S-adenosyl-l-methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs.