"Acceptor-donor-acceptor" motifs recognize the Watson-Crick, Hoogsteen and Sugar "donor-acceptor-donor" edges of adenine and adenosine-containing ligands

Nucleotides are among the most extensively exploited chemical moieties in nature and, as a part of a handful of different protein ligands, nucleotides play key roles in energy transduction, enzymatic catalysis and regulation of protein function. We have previously reported that in many proteins with different folds and functions a distinctive adenine-binding motif is involved in the recognition of the Watson-Crick edge of adenine. Here, we show that many proteins do have clear structural motifs that recognize adenosine (and some other nucleotides and nucleotide analogs) not only through the Watson-Crick edge, but also through the sugar and Hoogsteen edges. Each of the three edges of adenosine has a donor-acceptor-donor (DAD) pattern that is often recognized by proteins via a complementary acceptor-donor-acceptor (ADA) motif, whereby three distinct hydrogen bonds are formed: two conventional N-H...O and N-H...N hydrogen bonds, and one weak C-H...O hydrogen bond. The local conformation of the adenine-binding loop is betabetabeta or betabetaalpha and reflects the mode of nucleotide binding. Additionally, we report 21 proteins from five different folds that simultaneously recognize both the sugar edge and the Watson-Crick edge of adenine. In these proteins a unique beta-loop-beta supersecondary structure grasps an adenine-containing ligand between two identical adenine-binding motifs as part of the betaalphabeta-loop-beta fold.     

Structural features and hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a double helix containing two G.A mispairs

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5A resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-1 and O-6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).     

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.     

Recognition of nucleic acid bases and base-pairs by hydrogen bonding to amino acid side-chains

Sequence-specific protein-nucleic acid recognition is determined, in part, by hydrogen bonding interactions between amino acid side-chains and nucleotide bases. To examine the repertoire of possible interactions, we have calculated geometrically plausible arrangements in which amino acids hydrogen bond to unpaired bases, such as those found in RNA bulges and loops, or to the 53 possible RNA base-pairs. We find 32 possible interactions that involve two or more hydrogen bonds to the six unpaired bases (including protonated A and C), 17 of which have been observed. We find 186 "spanning" interactions to base-pairs in which the amino acid hydrogen bonds to both bases, in principle allowing particular base-pairs to be selectively targeted, and nine of these have been observed. Four calculated interactions span the Watson-Crick pairs and 15 span the G:U wobble pair, including two interesting arrangements with three hydrogen bonds to the Arg guanidinum group that have not yet been observed. The inherent donor-acceptor arrangements of the bases support many possible interactions to Asn (or Gln) and Ser (or Thr or Tyr), few interactions to Asp (or Glu) even though several already have been observed, and interactions to U (or T) only if the base is in an unpaired context, as also observed in several cases. This study highlights how complementary arrangements of donors and acceptors can contribute to base-specific recognition of RNA, predicts interactions not yet observed, and provides tools to analyze proposed contacts or design novel interactions.     

Structural Features and Hydration of d(C-G-C-G-A-A-T-T-A-G-C-G); a Double Helix Containing Two G.A Mispairs

Abstract

Single crystal X-ray diffraction techniques have been used to characterise the molecular structure of the title compound to 2.5Å resolution. The structure consists of ten standard Watson-Crick base pairs and two G.A mismatched base pairs. The purine-purine mismatches have guanine in the usual anti orientation with respect to the sugar and adenine in syn orientation. There are two hydrogen bonds formed between the mismatch bases, N-l and 0–6 of guanine with N-7 and N-6 of adenine respectively. The bulky purine-purine mismatches are accommodated with minor perturbation of the sugar-phosphate backbone. There is a slight improvement in base pair overlap at the mismatch sites. Details of the backbone conformation, base stacking interactions and hydration are presented and compared with those of the parent compound d(C-G-C-G-A-A-T-T-C-G-C-G).     

Theoretical studies on protein-nucleic acid interactions. II. Hydrogen bonding of amino acid side chains with bases and base pairs of nucleic acids

With a view to understanding the role of hydrogen bonds in the recognition of nucleic acids by proteins, hydrogen bonding between the bases and base pairs of nucleic acids and the amino acids (Asn, Gln, Asp and Glu, and charged residues Arg⁺, Glu^?, and Asp^?) has been studied by a second-order perturbation theory. Binding energies have been calculated for all possible configurations involving a pair of hydrogen bonds between the base (or base pair) and the amino acid residue. Our results show that the hydrogen bonding in these cases has a large contribution from electrostatic interaction. In general, the charged amino acids, compared to the uncharged ones, form more stable complexes with bases or base pairs. The hydrogen-bond energies are an order of magnitude smaller than the Coulombic interaction energies between basic amino acids (Lys⁺, Arg⁺, and His⁺) and the phosphate groups of nucleic acids. The stabilities of the complexes of amino acids Asn, Gln, Asp, and Glu with bases are in the order: G–X > C–X > A–X U–X or T–X, and G · C–X > A · T(U)–X, where X is one of these amino acid residues. It has been shown that Glu^? and Asp^? can recognize guanine in single-stranded nucleic acids; Arg⁺ can recognize G · C base pairs from A · T base pairs in double-stranded structures.     

Binding effects of Mn2+ and Zn2+ ions on the vibrational properties of guanine-cytosine base pairs in the Watson-Crick and Hoogsteen configurations

The binding effects of Mn(2+) and Zn(2+) ions on the vibrational properties of guanine-cytosine base pairs have been performed using density functional theory investigations. The calculations were carried out on Watson-Crick and Hoogsteen configurations of the base pairs. We have found, that in Watson-Crick configuration, the metal is coordinated to N7 atom of guanine while, in the case of Hoogsteen configuration, the coordination is at N3 atom of guanine. We have pointed out the vibrational bands that can be used to detect the presence of metallic ions in the Watson-Crick and Hoogsteen structures. Our results show that the vibrational amplitudes of metallic atoms are strong for wavenumbers lower than 600?cm(-1). Also, we predict that the distinction between Watson-Crick and Hoogsteen configurations can be seen around 85, 170 and 310?cm(-1).     

Direct identification of NH...N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy.

It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements.     

Non-planar structure of nitrous bases and non-coplanarity of Watson-Crick pairs.

We discuss the non-planar structural stability of the NH2-group in formamide, cytosine, adenine, guanine and aniline molecules. Based on the microwave data available on small amino derivatives and on the results of PCILO conformation study it is shown that the slope of the amino group HNH plane to the molecular plane in nitrous bases should be close to 40 degrees. One of the main consequences of the non-planar structure of bases is a comparatively large (approximately equal to 15 degrees) propeller twisting of purine and pyrimidine planes in the complementary adenine-thymine and guanine-cytosine pairs. It is concluded that the non-coplanarity of single Watson-Crick base pairs is their intrinsic property. The specificity of hydrogen bonding in pairs along with stacking is believed to be the original cause of their peculiar packing in crystals and in DNA and RNA structures.     

Sheared Aanti.Aanti base pairs in a destabilizing 2 x 2 internal loop: the NMR structure of 5'(rGGCAAGCCU)2

The 5'(rGGCAAGCCU)(2) duplex contains tandem A.A pairs. The three-dimensional structure of the 5'(rGGCAAGCCU)(2) duplex was modeled by molecular dynamics and energy minimization with NMR-derived distance and dihedral angle restraints. Although the 5'(rCAAG)(2) loop is thermodynamically destabilizing by 1.1 kcal/mol, the tandem A.A pairs adopt a predominant conformation: a sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment similar to that observed in the crystal structure of the P4-P6 domain of the Tetrahymena thermophila intron [Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685]. The NMR-derived structure of the 5'(rGGCAAGCCU)(2) duplex exhibits cross-strand hydrogen bonds from N3 of A4 to an amino hydrogen of A5 and from the 2' oxygen of the A4 sugar to the other amino hydrogen of A5. An intrastrand hydrogen bond is formed from the 2' OH hydrogen of A4 to O5' of A5. The cross-strand A5 bases are stacked. The Watson-Crick G-C regions are essentially A-form. The sheared anti-anti (A.A trans Hoogsteen/Sugar-edge) alignment provides potential contact sites for tertiary interactions and, therefore, is a possible target site for therapeutics. Thus, thermodynamically destabilizing internal loops can be preorganized for tertiary interactions or ligand binding.     

The structure of guanosine-thymidine mismatches in B-DNA at 2.5-A resolution

The structure of the deoxyoligomer d(C-G-C-G-A-A-T-T-T-G-C-G) was determined at 2.5-A resolution by single crystal x-ray diffraction techniques. The final R factor is 18% with the location of 71 water molecules. The oligomer crystallizes in a B-DNA-type conformation, with two strands interacting to form a dodecamer duplex. The double helix consists of four A X T and six G X C Watson-Crick base pairs and two G X T mismatches. The G X T pairs adopt a "wobble" structure with the thymine projecting into the major groove and the guanine into the minor groove. The mispairs are accommodated in the normal double helix by small adjustments in the conformation of the sugar phosphate backbone. A comparison with the isomorphous parent compound containing only Watson-Crick base pairs shows that any changes in the structure induced by the presence of G X T mispairs are highly localized. The global conformation of the duplex is conserved. The G X T mismatch has already been studied by x-ray techniques in A and Z helices where similar results were found. The geometry of the mispair is essentially identical in all structures so far examined, irrespective of the DNA conformation. The hydration is also similar with solvent molecules bridging the functional groups of the bases via hydrogen bonds. Hydration may be an important factor in stabilizing G X T mismatches. A characteristic of Watson-Crick paired A X T and G X C bases is the pseudo 2-fold symmetry axis in the plane of the base pairs. The G X T wobble base pair is pronouncedly asymmetric. This asymmetry, coupled with the disposition of functional groups in the major and minor grooves, provides a number of features which may contribute to the recognition of the mismatch by repair enzymes.     

Nuclear magnetic resonance study of hydrogen-bonded ring protons in oligonucleotide helices involving classical and nonclassical base pairs.

A study of the exchangeable ring nitrogen protons in aqueous solutions of oligonucleotide complexes involving Watson-Crick base pairs as well as Hoogsteen pairs and other nonclassical hydrogen bonding schemes shows that resolvable resonances in the low-field (-10 to -16 ppm from sodium 4,4-dimethyl-4-silapentanesulfonate) region can be detected in a variety of structures other than double stranded helices. Ring nitrogen proton resonances arising from the following hydrogen-bonding situations are reported: (1) AT and GC Watson-Crick base pairs in a self-complementary octanucleotide, dApApApGpCpTpTpT; (2) U-A-U base triples in complexes between oligo-U15 and AMP; (3) C-G-C+ base triples in complexes between oligo-C17 and GMP at acid pH; (4) s4U-A-s4U base triples in complexes between oligo-s4U15 and AMP, all of which involve both Watson-Crick and Hoogsteen base pairing to form triplexes; (5) C-C+ base pairing between protonated and unprotonated C residues in oligo-C17 at acid pH; and (6) I4 base quadruples in the four strand association among oligo-I at high salt. The behavior of the dA3G-CT3 helix is consistent with both fraying of the terminal base pairs and presence of intermediate states as the helix opens. In the monomer-oligomer complexes, under the conditions used here, the exchange appears to be governed by the dissociation rate of monomer from the complex. These findings suggest that those tertiary structure hydrogen bonds in tRNA involving ring nitrogen protons should have representative resonances in the low-field (11-16 ppm) proton NMR region in H2O.     

Dynamic properties of interaction between nucleic acid bases and models of amino acids

We have discussed the possible hydrogen bonding mode of adenine-uracil base dimers and peptide side chains, together with their characteristic effects on the stability of adenine-uracil base pairing in chloroform solution by using proton nuclear magnetic resonance methods, where acetamide and butyric acid were used as a model of residues of glutamine and glutamic acid, respectively, and were added to an adenine-uracil equimolar mixture. The stability of base pairing was affected significantly when the model compounds approached the adenine-uracil base pairs; that is, the Hoogsteen type was destroyed, while the Watson-Crick type was formed more favorably.     

Molecular recognition in purine-rich internal loops: thermodynamic,structural, and dynamic consequences of purine for adenine substitutions in 5'(rGGCAAGCCU)2

The contribution of amino groups to the thermodynamics, structure, and dynamics of tandem A.A mismatches is investigated by substitution of purine (P) for adenine (A) within the RNA duplex, 5'(rGGCAAGCCU)(2), to give 5'(rGGCPAGCCU)(2), 5'(rGGCAPGCCU)(2), and 5'(rGGCPPGCCU)(2). The 5'(rGGCAAGCCU)(2) duplex has sheared A(anti).A(anti) (A.A trans Hoogsteen/Sugar-edge) pairs in which the A5 amino group is involved in hydrogen bonds but the A4 amino group is not [Znosko, B. M., Burkard, M. E., Schroeder, S. J., Krugh, T. R., and Turner, D. H. (2002) Biochemistry 41, 14969-14977]. In comparison to 5'(rGGCAAGCCU)(2), replacing the amino group of A4 with a hydrogen stabilizes the duplex by 1.3 kcal/mol, replacement of the A5 amino group destabilizes the duplex by 0.6 kcal/mol, and replacement of both A4 and A5 amino groups destabilizes the duplex by 0.8 kcal/mol. In NMR structures, the P.A noncanonical pairs of the 5'(rGGCPAGCCU)(2) duplex have a sheared anti-anti structure (P.A trans Hoogsteen/Sugar-edge) with P4.A5 interstrand hydrogen bonding and A5 bases that interstrand stack, similar to the structure of 5'(rGGCAAGCCU)(2). In contrast, the A.P pairs of the 5'(rGGCAPGCCU)(2) duplex have a face-to-face conformation (A.P trans Watson-Crick/Watson-Crick) with intrastrand stacking resembling typical A-form geometry. Although the P5 bases in 5'(rGGCPPGCCU)(2) are involved in an interstrand stack, the loop region is largely undefined. The results illustrate that both hydrogen-bonded and non-hydrogen-bonded amino groups play important roles in determining the thermodynamic, structural, and dynamic characteristics of purine rich internal loops.     

Interactions of quinoxaline antibiotic and DNA: the molecular structure of a triostin A-d(GCGTACGC) complex

The crystal structure of a DNA octamer d(GCGTACGC) complexed to an antitumor antibiotic, triostin A, has been solved and refined to 2.2 A resolution by x-ray diffraction analysis. The antibiotic molecule acts as a true bis intercalator surrounding the d(CpG) sequence at either end of the unwound right-handed DNA double helix. As previously observed in the structure of triostin A-d(CGTACG) complex (A.H.-J. Wang, et. al., Science, 225, 1115-1121 (1984)), the alanine amino acid residues of the drug molecule form sequence-specific hydrogen bonds to guanines in the minor groove. The two central A.T base pairs are in Hoogsteen configuration with adenine in the syn conformation. In addition, the two terminal G.C base pairs flanking the quinoxaline rings are also held together by Hoogsteen base pairing. This is the first observation in an oligonucleotide of. Hoogsteen G.C base pairs where the cytosine is protonated. The principal functional components of a bis-intercalative compound are discussed.     

Destabilization of DNA Guanine Quadruplex Structure by Foldback Triplex-Forming Oligodeoxynucleotides

Abstract

An oligodeoxynucleotide designed to bind to a single stranded guanine rich DNA sequence through Watson-Crick followed by Hoogsteen hydrogen bonds was found to destabilize quadruplex structure formed by the target sequence. However, the conventional antisense and antigene oligonucleotides were unable to destabilize the same quadruplex structure.     

Structure of DNA polymerase beta with the mutagenic DNA lesion 8-oxodeoxyguanine reveals structural insights into its coding potential

Oxidative damage to DNA generates 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG). During DNA replication and repair synthesis, 8-oxodG can pair with cytosine or adenine. The ability to accurately replicate through this lesion depends on the DNA polymerase. We report the first structure of a polymerase with a promutagenic DNA lesion, 8-oxodG, in the confines of its active site. The modified guanine residue is in an anti conformation and forms Watson-Crick hydrogen bonds with an incoming dCTP. To accommodate the oxygen at C8, the 5'-phosphate backbone of the templating nucleotide flips 180 degrees. Thus, the flexibility of the template sugar-phosphate backbone near the polymerase active site is one parameter that influences the anti-syn equilibrium of 8-oxodG. Our results provide insights into the mechanisms employed by polymerases to select the complementary dNTP.     

Interactions of Quinoxaline Antibiotic and DNA.: The Molecular Structure of a Triostin A—d(GCGTACGC) Complex

Abstract

The crystal structure of a DNA. octamer d(GCGTA.CGC) complexed to an antitumor antibiotic, triostin A, has been solved and refined to 2.2 Å resolution by x-ray diffraction analysis. The antibiotic molecule acts as a true bis intercalator surrouding the d(CpG) sequence at either end of the unwound right-handed DNA. double helix. A.s previously observed in the structure of triostin A.—d(CGTA.CG) complex (A.H.-J. Wang, et. al., Science, 225,1115–1121 (1984)), the alanine amino acid residues of the drug molecule form sequence-specific hydrogen bonds to guanines in the minor groove. The two central A · T base pairs are in Hoogsteen configuration with adenine in the syn conformation. In addition, the two terminal G · C base pairs flanking the quinoxaline rings are also held together by Hoogsteen base pairing. This is the first observation in an oligonucleotide of. Hoogsteen G · C base pairs where the cytosine is protonated. The principal functional components of a bis-intercalative compound are discussed.