Frequent occurrence of the T-loop RNA folding motif in ribosomal RNAs

Analysis of atomic resolution structures of the rRNAs within the context of the 50S and the 30S ribosomal subunits have revealed the presence of nine examples of a recurrent structural motif, first observed in the TpsiC loop of tRNAs. The key component of this T-loop motif is a UA trans Watson-Crick/Hoogsteen base pair stacked on a Watson-Crick pair on one side. This motif is stabilized by several noncanonical hydrogen bonds, facilitating RNA-RNA as well as RNA-protein interactions. In particular, the sugar edge of the purine on the 3' side of the pivotal uridine in the UA pair frequently forms a noncanonical base pair with a distant residue. The bulged-out bases, usually seen as part of the motif, also use their Watson-Crick edges to interact with nearby residues via base-specific hydrogen bonds. In certain occurrences, a backbone reversal is stabilized by specific hydrogen bonds as is observed in the U-turn motifs and the adenosine residue of the key UA pair interacts with a third base via its Watson-Crick edge, essentially generating a base triple.     

Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes

Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.     

A two-stranded template-based approach to G.(C-A) triad formation: designing novel structural elements into an existing DNA framework

We have designed a DNA sequence, d(G-G-G-T-T-C-A-G-G), which dimerizes to form a 2-fold symmetric G-quadruplex in which G(syn). G(anti).G(syn).G(anti) tetrads are sandwiched between all trans G. (C-A) triads. The NMR-based solution structural analysis was greatly aided by monitoring hydrogen bond alignments across N-H...N and N-H...O==C hydrogen bonds within the triad and tetrad, in a uniformly ((13)C,(15)N)-labeled sample of the d(G-G-G-T-T-C-A-G-G) sequence. The solution structure establishes that the guanine base-pairs with the cytosine through Watson-Crick G.C pair formation and with adenine through sheared G.A mismatch formation within the G.(C-A) triad. A model of triad DNA was constructed that contains the experimentally determined G.(C-A) triad alignment as the repeating stacked unit.     

Estimation of strength in different extra Watson-Crick hydrogen bonds in DNA double helices through quantum chemical studies

It was shown earlier, from database analysis, model building studies, and molecular dynamics simulations that formation of cross-strand bifurcated or Extra Watson-Crick hydrogen (EWC) bonds between successive base pairs may lead to extra rigidity to DNA double helices of certain sequences. The strengths of these hydrogen bonds are debatable, however, as they do not have standard linear geometry criterion. We have therefore carried out detailed ab initio quantum chemical studies using RHF/6-31G(2d,2p) and B3LYP/6-31G(2p,2d) basis sets to determine strengths of several bent hydrogen bonds with different donor and acceptors. Interaction energy calculations, corrected for the basis set superposition errors, suggest that N-H...O type bent EWC hydrogen bonds are possible along same strands or across the strands between successive base pairs, leading to significant stability (ca. 4-9 kcal/mol). The N-H...N and C-H...O type interactions, however, are not so stabilizing. Hence, consideration of EWC N-H...O H-bonds can lead to a better understanding of DNA sequence directed structural features.     

Observation of a unique pattern of bifurcated hydrogen bonds in the crystal structures of the N-glycoprotein linkage region models

Elucidation of the intra- and intermolecular carbohydrate-protein interactions would greatly contribute toward obtaining a better understanding of the structure-function correlations of the protein-linked glycans. The weak interactions involving C-H...O have recently been attracting immense attention in the domain of biomolecular recognition. However, there has been no report so far on the occurrence of C-H...O hydrogen bonds in the crystal structures of models and analogs of N-glycoproteins. We present herein an analysis of C-H...O interactions in the crystal structures of all N-glycoprotein linkage region models and analogs. The study reveals a cooperative network of bifurcated hydrogen bonds consisting of N-H...O and C-H...O interactions seen uniquely for the models. The cooperative network consists of two antiparallel chains of bifurcated hydrogen bonds, one involving N1-H, C2'-H and O1' of the aglycon moiety and the other involving N2-H, C1-H and O1' of the sugar. Such bifurcated hydrogen bonds between the core glycan and protein are likely to play an important role in the folding and stabilization of proteins.     

GXXXG and GXXXA motifs stabilize FAD and NAD(P)-binding Rossmann folds through C(alpha)-H... O hydrogen bonds and van der waals interactions

Here we present evidence that domains in soluble proteins containing either the GXXXG or GXXXA motif are stabilized by the interaction of a beta-strand with the following alpha-helix. As an example, we characterized a beta-strand-helix interaction from the FAD or NAD(P)-binding Rossmann fold. The Rossmann fold is one of the three most highly represented folds in the Protein Data Bank (PDB). A subset of the proteins that adopt the Rossmann fold also bind to nucleotide cofactors such as FAD and NAD(P) and function as oxidoreductases. These Rossmann folds can often be identified by the short amino acid sequence motif, GX(1-2)GXXG. Here, we present evidence that in addition to this sequence motif, Rossmann folds that bind FAD and NAD(P) also typically contain either GXXXG or GXXXA motifs, where the first glycyl residue of these motifs and the third glycyl residue of the GX(1-2)GXXG motif are the same residue. These two motifs appear to stabilize the Rossmann fold: the first glycyl residue of either the GXXXG or GXXXA motif contacts the carbonyl oxygen atom from the first glycyl residue of the GX(1-2)GXXG motif consistent with the formation of a C(alpha)-H cdots, three dots, centered O hydrogen bond. In addition, both the glycyl and alanyl residues of the GXXXG or GXXXA motifs form van der Waals interactions with either a valine or isoleucine residue located either seven or eight residues further back along the polypeptide chain from the first glycine of the GXXXG or GXXXA motifs. Therefore, we combine both the GX(1-2)GXXG and GXXXG/A motifs into an extended motif, V/IXGX(1-2)GXXGXXXG/A, that is more strongly indicative than previously described motifs of Rossmann folds that bind FAD or NAD(P). The V/IXGX(1-2)GXXGXXXG/A motif can be used to search genomic sequence data and to annotate the function of proteins containing the motif as oxidoreductases, including proteins of previously unknown function.     

Registering alpha-helices and beta-strands using backbone C-H...O interactions

The possible occurrence of a novel helix terminating structural motif in proteins involving a stabilizing short C-H...O interaction has been examined using a dataset of 634 non-homologous protein structures (相似文献   

Study of the ATP-binding site of helicase IV from Escherichia coli

Helicases contain conserved motifs involved in ATP/magnesium/nucleic acid binding and in the mechanisms coupling nucleotide hydrolysis to duplex unwinding. None of these motifs are located at the adenine-binding pocket of the protein. We show here that the superfamily I helicase, helicase IV from Escherichia coli, utilizes a conserved glutamine and conserved aromatic residue to interact with ATP. Other superfamily I helicases such as, UvrD/Rep/PcrA also possess these residues but in addition they interact with adenine via a conserved arginine, which is replaced by a serine in helicase IV. Mutation of this serine residue in helicase IV into histidine or methionine leads to proteins with unaffected ATPase and DNA-binding activities but with low helicase activity. This suggests that residues located at the adenine-binding pocket, in addition to be involved in ATP-binding, are important for efficient coupling between ATP hydrolysis and DNA unwinding.     

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi apparatus vesicles

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3'-phosphate 5'-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3'-phosphate 5'-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.     

Requirement of Watson-Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta

Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase eta (Poleta) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Poleta are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Poleta; in this regard, Poleta differs strikingly from classical high-fidelity DNA polymerases.     

De novo design and characterization of a helical hairpin eicosapeptide; emergence of an anion receptor in the linker region

De novo design of supersecondary structures is expected to provide useful molecular frameworks for the incorporation of functional sites as in proteins. A 21 residue long, dehydrophenylalanine-containing peptide has been de novo designed and its crystal structure determined. The apolar peptide folds into a helical hairpin supersecondary structure with two right-handed helices, connected by a tetraglycine linker. The helices of the hairpin interact with each other through a combination of C-H.O and N-H.O hydrogen bonds. The folding of the apolar peptide has been realized without the help of either metal ions or disulphide bonds. A remarkable feature of the peptide is the unanticipated occurrence of an anion binding motif in the linker region, strikingly similar in conformation and function to the "nest" motif seen in several proteins. The observation supports the view for the possible emergence of rudimentary functions over short sequence stretches in the early peptides under prebiotic conditions.     

Effect of nucleotides on translocation of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi apparatus vesicles

Recent studies from this laboratory have suggested that rat-liver Golgi apparatus derived membranes contain different proteins which can translocate in vitro CMP-N-acetylneuraminic acid, GDP-fucose and adenosine 3′-phosphate 5′-phosphosulfate from an external compartment into a lumenal one. The aim of this study was to define the role of the nucleotide, sugar and sulfate moieties of sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate in translocation of these latter compounds across Golgi vesicle membranes. Indirect evidence was obtained suggesting that the nucleotide (but not sugar or sulfate) is a necessary recognition feature for binding to the Golgi membrane (measured as inhibition of translocation) but is not sufficient for overall translocation; this latter event also depends on the type of sugar. Important recognition features for inhibition of translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were found to be the type of nucleotide base (purine or pyrimidine) and the position of the phosphate group in the ribose. Thus, UMP and CMP were found to be competitive inhibitors of translocation of CMP-N-acetylneuraminic acid, while AMP did not inhibit. Structural features of the nucleotides which were less important in inhibition of translocation (and thus presumably in binding) of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate were the number of phosphate groups in the nucleotide (CDP and CMP inhibited to a similar extent), the presence of ribose or deoxyribose in the nucleotide, a replacement of hydrogen in positions 5 of pyrimidines or 8 in purines by halogens or an azido group. The sugar or sulfate did not inhibit translocation of the above sugar nucleotides and adenosine 3′-phosphate 5′-phosphosulfate into Golgi vesicles and therefore appear not to be involved in their binding to the Golgi membrane.     

Effect of adenosine on the adenine nucleotide content and metabolism of hepatocytes.

ADENOSINE (0.5 MM) added to hepatocyte suspensions increased the intracellular concentration of ATP and total adenine nucleotides within 60 min up to three-fold. 2. Adenosine at 0.5 mM inhibited gluconeogenesis from lactate by about 50%. At higher adenosine concentrations the inhibition was less. There was no strict parallelism between the time-course of the increase of the adenine nucleotide content and the time-course of the inhibition of gluconeogenesis from lactate. 3. Adenosine abolished the accelerating effects of oleate and dibutyryl cyclic AMP on gluconeogenesis from lactate. 4. Gluconeogenesis was no significant effect of adenosine with fructose, dihydroxyacetone or glycerol. With asparagine, adenosine caused anacceleration of glucose formation. 5. Adenosine incorporation into adenine nucleotides accounted for about 20% of the adenosine removal. 6. Inosine, hypoxanthine or adenine compared with adenosine gave relatively slight increases of adenine nucleotides. 7. Urea synthesis from NH4Cl under optimum conditions i.e. in the presence of ornithine, lactate and oleate, was also inhibited by adenosine. The inhibition increased with the adenosine concentration and was 65% at 4 mM-adenosine. Again there was no correlation between the degree of inhibition of urea synthesis and the increase in the adenine nucleotide content. 8. The basal O2 consumption, the increased O2 consumption on the addition of oleate and the rate of formation of ketone bodies were not affected by the addition of adenosine. The [beta-hydroxybutyrate]/[acetoacetate] ratio was increased by adenosine, provided that lactate was present. 9. The increase of the adenine nucleotide content of the hepatocytes on the addition of adenosine may be explained on the assumption that adenosine kinase is not regulated by feedback but by substrate supply.     

GXXXG and AXXXA: common alpha-helical interaction motifs in proteins, particularly in extremophiles

The GXXXG motif is a frequently occurring sequence of residues that is known to favor helix-helix interactions in membrane proteins. Here we show that the GXXXG motif is also prevalent in soluble proteins whose structures have been determined. Some 152 proteins from a non-redundant PDB set contain at least one alpha-helix with the GXXXG motif, 41 +/- 9% more than expected if glycine residues were uniformly distributed in those alpha-helices. More than 50% of the GXXXG-containing alpha-helices participate in helix-helix interactions. In fact, 26 of those helix-helix interactions are structurally similar to the helix-helix interaction of the glycophorin A dimer, where two transmembrane helices associate to form a dimer stabilized by the GXXXG motif. As for the glycophorin A structure, we find backbone-to-backbone atomic contacts of the C alpha-H...O type in each of these 26 helix-helix interactions that display the stereochemical hallmarks of hydrogen bond formation. These glycophorin A-like helix-helix interactions are enriched in the general set of helix-helix interactions containing the GXXXG motif, suggesting that the inferred C alpha-H...O hydrogen bonds stabilize the helix-helix interactions. In addition to the GXXXG motif, some 808 proteins from the non-redundant PDB set contain at least one alpha-helix with the AXXXA motif (30 +/- 3% greater than expected). Both the GXXXG and AXXXA motifs occur frequently in predicted alpha-helices from 24 fully sequenced genomes. Occurrence of the AXXXA motif is enhanced to a greater extent in thermophiles than in mesophiles, suggesting that helical interaction based on the AXXXA motif may be a common mechanism of thermostability in protein structures. We conclude that the GXXXG sequence motif stabilizes helix-helix interactions in proteins, and that the AXXXA sequence motif also stabilizes the folded state of proteins.     

Structural basis of DNA flexibility

It has been recently shown by us, on the basis of crystal structure database that the flexibility of B-DNA double helices depends significantly on their base sequence. Our model building studies further indicated that the existence of bifurcated cross-strand hydrogen bonds between successive base pairs is possibly the main factor behind the sequence directed DNA flexibility. These cross-strand hydrogen bonds are, of course, weaker than the usual Watson-Crick hydrogen bonds and their bond geometry is characterized by relatively larger bond lengths and smaller bond angles. We have tried to improve our model structures by incorporating non-planarity of the amino groups in DNA bases due to the presence of lone pair electrons at the nitrogen atoms. Energy minimization studies have been carried out by using different quantum chemical methods, whereby it is found that in all cases of N-H....O type cross-strand hydrogen bonds, the bond geometry improves significantly. In the cases of N-H....N type hydrogen bonds, however, no such consistent improvements can be noticed. Perhaps the true picture would emerge only if all the other interactions present in the DNA macromolecule could be appropriately taken into account.     

N-H...O, O-H...O, and C-H...O hydrogen bonds in protein-ligand complexes: strong and weak interactions in molecular recognition

The characteristics of N-H...O, O-H...O, and C-H...O hydrogen bonds are examined in a group of 28 high-resolution crystal structures of protein-ligand complexes from the Protein Data Bank and compared with interactions found in small-molecule crystal structures from the Cambridge Structural Database. It is found that both strong and weak hydrogen bonds are involved in ligand binding. Because of the prevalence of multifurcation, the restrictive geometrical criteria set up for hydrogen bonds in small-molecule crystal structures may need to be relaxed in macromolecular structures. For example, there are definite deviations from linearity for the hydrogen bonds in protein-ligand complexes. The formation of C-H...O hydrogen bonds is influenced by the activation of the C(alpha)-H atoms and by the flexibility of the side-chain atoms. In contrast to small-molecule structures, anticooperative geometries are common in the macromolecular structures studied here, and there is a gradual lengthening as the extent of furcation increases. C-H...O bonds formed by Gly, Phe, and Tyr residues are noteworthy. The numbers of hydrogen bond donors and acceptors agree with Lipinski's "rule of five" that predicts drug-like properties. Hydrogen bonds formed by water are also seen to be relevant in ligand binding. Ligand C-H...O(w) interactions are abundant when compared to N-H...O(w) and O-H...O(w). This suggests that ligands prefer to use their stronger hydrogen bond capabilities for use with the protein residues, leaving the weaker interactions to bind with water. In summary, the interplay between strong and weak interactions in ligand binding possibly leads to a satisfactory enthalpy-entropy balance. The implications of these results to crystallographic refinement and molecular dynamics software are discussed.     

Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins

Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (O2-) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced O2- formation induced by submaximally and maximally stimulatory concentrations of fMet-Leu-Phe up to fivefold. On the other hand, FMet-Leu-Phe primed neutrophils to generate O2- upon exposure to nucleotides. At a concentration of 100 microM, purine nucleotides enhanced O2- formation in the effectiveness order adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]) greater than ITP greater than guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) greater than ATP = adenosine 5'-O-[2-thio]triphosphate (Sp-diastereomer) = GTP = guanosine 5'-O-[2-thio]diphosphate (GDP[beta S] = ADP greater than adenosine 5'-[beta, gamma-imido]triphosphate = adenosine 5'-O-[2-thio]triphosphate] (Rp-diastereomer). Pyrimidine nucleotides stimulated fMet-Leu-Phe-induced O2- formation in the effectiveness order uridine 5'-O-[3-thio]triphosphate (UTP[gamma S]) = UTP greater than CTP. Uracil (UDP[beta S]) = uridine 5'-O[2-thio]triphosphate (Rp-diastereomer) (Rp)-UTP[beta S]) = UTP greater than CTP. Uracil nucleotides were similarly effective potentiators of O2- formation as the corresponding adenine nucleotides. GDP[beta S] and UDP[beta S] synergistically enhanced the stimulatory effects of ATP[gamma S], GTP[gamma S] and UTP[gamma S]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated fMet-Leu-Phe-induced release of beta-glucuronidase with similar nucleotide specificities as for O2- formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine-nucleotide-binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation.