NMR study of a novel RNA quadruplex structure.

The structure of an RNA oligomer, r (GGAGGUUUUGGAGG) (R14-2) whose G-G steps are separated by adenine and uracil residues has been investigated by NMR. In the presence of 20 mM K+, a novel dimeric multiplex architecture is adopted by two strands of R14-2. In each strand a UUUU loop and two A residues connect four parallel G-G steps that pair-align into two tetrads. One of the tetrads is further pair-aligned by two A residues through the sheared mismatch and a novel hexad is subsequently formed. Two hexads coming from two different strands stack to make a dimeric multiplex. All of the guanosine and adenosine residues take an anti conformation.     

NMR studies of fully modified locked nucleic acid (LNA) hybrids: solution structure of an LNA:RNA hybrid and characterization of an LNA:DNA hybrid

LNA is a bicyclic nucleic acid analogue that contains one or more 2'-O,4'-C methylene linkage(s), which effectively locks the furanose ring in a C3'-endo conformation. We report here the NMR solution structure of a nonamer LNA:RNA hybrid and a structural characterization of a nonamer LNA:DNA hybrid, where the LNA strands are composed entirely of LNA nucleotides. This is the first structural characterization of fully modified LNA oligonucleotides. The high-resolution structure reveals that the LNA:RNA hybrid adopts an almost canonical A-type duplex morphology. The helix axis is almost straight and the duplex geometry is regular. This shows that fully modified LNA oligomers can hybridize with complementary RNA and form duplexes within the Watson-Crick framework. The LNA:DNA hybrid structurally resembles an RNA:DNA hybrid as shown by determination of deoxyribose sugar puckers and analysis of NOESY NMR spectra.     

NMR observation of T-tetrads in a parallel stranded DNA quadruplex formed by Saccharomyces cerevisiae telomere repeats.

We report here the NMR structure of the DNA sequence d-TGGTGGC containing two repeats of Saccharomyces cerevisiae telomere DNA which is unique in that it has a single thymine in the repeat sequence and the number of Gs can vary from one to three. The structure is a novel quadruplex incor-porating T-tetrads formed by symmetrical pairing of four Ts via O4-H3 H-bonds in a plane. This is in contrast to the previous results on other telomeric sequences which contained more than one T in the repeat sequences and they were seen mostly in the flexible regions of the structures. We observed that the T4-tetrad was nicely accommodated in the center of the G-quadruplex, but it caused a small underwinding of the right handed helix. The T tetrad stacked well on the adjacent G3-tetrad, but poorly on the G5 tetrad. Likewise, T1 also formed a stable T-tetrad at the 5' end of the quadruplex. To our knowledge, this is the first report of T-tetrad formation in DNA structures. These observations are of significance from the points of view of both structural diversity and specific recognitions.     

NMR solution structures of LNA (locked nucleic acid) modified quadruplexes

We have determined the NMR solution structures of the quadruplexes formed by d(TGLGLT) and d(TL₄T), where L denotes LNA (locked nucleic acid) modified G-residues. Both structures are tetrameric, parallel and right-handed and the native global fold of the corresponding DNA quadruplex is retained upon introduction of the LNA nucleotides. However, local structural alterations are observed owing to the locked LNA sugars. In particular, a distinct change in the sugar–phosphate backbone is observed at the G2pL3 and L2pL3 base steps and sequence dependent changes in the twist between tetrads are also seen. Both the LNA modified quadruplexes have raised thermostability as compared to the DNA quadruplex. The quadruplex-forming capability of d(TGLGLT) is of particular interest as it expands the design flexibility for stable parallel LNA quadruplexes and shows that LNA nucleotides can be mixed with DNA or other modified nucleic acids. As such, LNA-based quadruplexes can be decorated by a variety of chemical modifications. Such LNA quadruplex scaffolds might find applications in the developing field of nanobiotechnology.     

NMR solution structure of butantoxin

The NMR structure of a new toxin, butantoxin (BuTX), which is present in the venoms of the three Brazilian scorpions Tityus serrulatus, Tityus bahiensis, and Tityus stigmurus, has been investigated. This toxin was shown to reversibly block the Shaker B potassium channels (K(d) approximately 660 nM) and inhibit the proliferation of T-cells and the interleukin-2 production of antigen-stimulated T-helper cells. BuTX is a 40 amino acid basic protein stabilized by the four disulfide bridges: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36, and Cys20-Cys38. The latter three are conserved among all members of the short-chain scorpion toxin family, while the first is unique to BuTX. The three-dimensional structure of BuTX was determined using (1)H-NMR spectroscopy. NOESY, phase sensitive COSY (PH-COSY), and amide hydrogen exchange data were used to generate constraints for molecular modeling calculations. Distance geometry and simulated annealing calculations were performed to generate a family of 49 structures free of constraint violations. The secondary structure of BuTX consists of a short 2(1/2) turn alpha-helix (Glu15-Phe23) and a beta-sheet. The beta-sheet is composed of two well-defined antiparallel strands (Gly29-Met32 and Lys35-Cys38) connected by a type-I' beta-turn (Asn33-Asn34). Residues Cys5-Ala9 form a quasi-third strand of the beta-sheet. The N-terminal C2-C5 disulfide bridge unique to this toxin does not appear to confer stability to the protein.     

The solution structure of a locked nucleic acid (LNA) hybridized to DNA

LNA (Locked Nucleic Acids) is a novel oligonucleotide analogue containing a conformationally restricted nucleotide with a 2'-O, 4'-C-methylene bridge that induces unprecedented thermal affinities when mixed with complementary single stranded DNA and RNA. We have used two-dimensional 1H NMR spectroscopy obtained at 750 and 500 MHz to determine a high resolution solution structure of an LNA oligonucleotide hybridized to the complementary DNA strand. The determination of the structure was based on a complete relaxation matrix analysis of the NOESY cross peaks followed by restrained molecular dynamics calculations. Forty final structures were generated for the duplex from A-type and B-type dsDNA starting structures. The root-mean-square deviation (RMSD) of the coordinates for the forty structures of the complex was 0.32A. The structures were analysed by use of calculated helix parameters. This showed that the values for rise and buckle in the LNA duplex is markedly different from canonical B-DNA at the modification site. A value of twist similar to A-DNA is also observed at the modification site. The overall length of the helix which is 27.3 A. The average twist over the sequence are 35.9 degrees +/- 0.3 degrees. Consequently, the modification does not cause the helix to unwind. The bis-intercalation of the thiazole orange dye TOTO to the LNA duplex was also investigated by 1H NMR spectroscopy to sense the structural change from the unmodified oligonucleotide. We observed that the bis-intercalation of TOTO is much less favourable in the 5'-CT(L)AG-3' site than in the unmodified 5'-CTAG-3' site. This was related to the change in the base stacking of the LNA duplex compared to the unmodified duplex.     

NMR solution structure determination of RNAs

During the past several years, there have been significant advances in NMR solution structure determination of macromolecules. The ability to easily measure residual dipolar couplings, to directly detect NHellipsisN hydrogen bonding interactions and to study much larger macromolecules by the application of heteronuclear experiments that select narrow lines in 2D and 3D spectra of isotopically labeled molecules promises to dramatically improve solution structure determination of nucleic acids.     

High-temperature solution NMR structure of TmCsp

Cold shock proteins (Csps) are assumed to play a central role in the regulation of gene expression under cold shock conditions. Acting as single-stranded nucleic acid-binding proteins, they trigger the translation process and are therefore involved in the compensation of the influence of low temperatures (cold shock) upon the cell metabolism. However, it is unknown so far how Csps are switched on and off as a function of temperature. The aim of the present study is the study of possible structural changes responsible for this switching process. (1)H-(15)N HSQC spectra recorded at different temperatures and chemical-shift analysis have indicated subtle conformational changes for the cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima (TmCsp) when the temperature is elevated from 303 K to its physiological temperature (343 K). The three-dimensional structure of TmCsp was determined by nuclear magnetic resonance (NMR) spectroscopy at 343 K to obtain quantitative information concerning these structural changes. By use of residual dipolar couplings, the loss of NOE information at high temperature could be compensated successfully. Most pronounced conformational changes compared with room-temperature conditions are observed for amino acid residues closely neighbored to two characteristic beta-bulges and a well-defined loop region of the protein. Because the residues shown to be responsible for the interaction of TmCsp with single-stranded nucleic acids can almost exclusively be found within these regions, nucleic acid-binding activity might be down-regulated with increasing temperature by the described conformational changes.     

HIV-integrase aptamer folds into a parallel quadruplex: a thermodynamic study

Short guanine-rich sequences have a tendency to form quadruplexes that are stabilized by G-quartets with specific cation coordination. Quadruplexes are part of telomeres at the ends of chromosomes and play an important role in the regulation of gene expression. In addition, there is a strong interest in the therapeutic and biotechnological potential of quadruplex oligonucleotides. The HIV-integrase aptamer, d(GGGT)(4), demonstrated unusually favorable van't Hoff thermodynamics, and based on NMR studies the aptamer was proposed to fold into an antiparallel structure. Here we probed an apparent discrepancy between the NMR structure and the quadruplex topology suggested by circular dichroism (CD). Systematic thermodynamic analyses of d(GGGT)(4) and variants containing sequence modifications or missing specific nucleotides are consistent with a parallel quadruplex fold. CD studies carried out over a wide concentration range did not support a possible structural transition upon increasing strand concentration. Taken together, both optical and thermodynamic studies performed here strongly support a parallel fold for the d(GGGT)(4) aptamer.     

The NMR solution structure of recombinant RGD-hirudin

The solution structure of a new recombinant RGD-hirudin, which has the activities of anti-thrombin and anti-platelet aggregation, was determined by (1)H nuclear magnetic resonance spectroscopy and compared with the conformations of recombinant wild-type hirudin and hirudin (variant 2, Lys47) of the hirudin thrombin complex. On the basis of total 1284 distance and dihedral angle constraints derived from a series of NMR spectra, 20 conformers were computed with ARIA/CNS programs. The structure of residues 3-30 and 37-48 form a molecular core with two antiparallel beta-sheets as the other two hirudins. However, significant differences were found in the surface electrostatic charge distributions among the three hirudins, especially in the RGD segment of recombinant RGD-hirudin. This difference may be greatly beneficial to its additional function of anti-platelet aggregation. The difference in extended C-terminal makes its both ionic and hydrophobic interactions with the fibrinogen recognition exosite of thrombin more effective.     

Studies on solution NMR structure of brazzein——Secondary structure and molecular scaffold

Brazzein is a sweet-tasting protein isolated from the fruit of West African plantPentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far and is highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300 K) is presented. The complete sequence specific assignments of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one alpha-helix (residues 21-29), one short 3(10)-helix (residues 14-17), two strands of antiparallel beta-sheet (residues 34-39, 44-50) and probably a third strand (residues 5-7) near the N-terminus. A comparative analysis found that brazzein shares a so-called 'cysteine-stabilized alpha-beta' (CSalphabeta) motif with scorpion neurotoxins, insect defensins and plant gamma - thionins. The significance of this multi-function motif, the possible active sites and the structural basis of themostability were discussed.     

Studies on solution NMR structure of brazzein

Brazzein is a sweet-tasting protein isolated from the fruit of West African plantPentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far and is highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300 K) is presented. The complete sequence specific assignments of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one α-helix (residues 21–29), one short 3₁₀-helix (residues 14–17), two strands of antiparallel β-sheet (residues 34–39, 44–50) and probably a third strand (residues 5–7) near the N-terminus. A comparative analysis found that brazzein shares a so-called ‘cysteine-stabilized alpha-beta’ (CSαβ) motif with scorpion neurotoxins, insect defensins and plant γ - thionins. The significance of this multi-function motif, the possible active sites and the structural basis of themostability were discussed.     

NMR methods for studying quadruplex nucleic acids

Solution NMR spectroscopy has traditionally played a central role in examining quadruplex structure, dynamics, and interactions. Here, an overview is given of the methods currently applied to structural, dynamics, thermodynamics, and kinetics studies of nucleic acid quadruplexes and associated cations.     

Solution structure of a dsDNA:LNA triplex

We have determined the NMR structure of an intramolecular dsDNA:LNA triplex, where the LNA strand is composed of alternating LNA and DNA nucleotides. The LNA oligonucleotide binds to the dsDNA duplex in the major groove by formation of Hoogsteen hydrogen bonds to the purine strand of the duplex. The structure of the dsDNA duplex is changed to accommodate the LNA strand, and it adopts a geometry intermediate between A- and B-type. There is a substantial propeller twist between base-paired nucleobases. This propeller twist and a concomitant large propeller twist between the purine and LNA strands allows the pyrimidines of the LNA strand to interact with the 5′-flanking duplex pyrimidines. Altogether, the triplex has a regular global geometry as shown by a straight helix axis. This shows that even though the third strand is composed of alternating DNA and LNA monomers with different sugar puckers, it forms a seamless triplex. The thermostability of the triplex is increased by 19°C relative to the unmodified DNA triplex at acidic pH. Using NMR spectroscopy, we show that the dsDNA:LNA triplex is stable at pH 8, and that the triplex structure is identical to the structure determined at pH 5.1.     

The high-resolution crystal structure of a parallel intermolecular DNA G-4 quadruplex/drug complex employing syn glycosyl linkages

We have determined the X-ray structure of the complex between the DNA quadruplex d(5′-GGGG-3′)₄ and daunomycin, as a potential model for studying drug–telomere interactions. The structure was solved at 1.08 Å by direct methods in space group I4. The asymmetric unit comprises a linear arrangement of one d(GGGG) strand, four daunomycin molecules, a second d(GGGG) strand facing in the opposite direction to the first, and Na and Mg cations. The crystallographic 4-fold axis generates the biological unit, which is a 12-layered structure comprising two sets of four guanine layers, with four layers each of four daunomycins stacked between the 5′ faces of the two quadruplexes. The daunomycin layers fall into two groups which are novel in their mode of self assembly. The only contacts between daunomycin molecules within any one of these layers are van der Waals interactions, however there is substantial π–π stacking between successive daunomycin layers and also with adjacent guanine layers. The structure differs significantly from all other parallel d(TGGGGT)₄ quadruplexes in that the 5′ guanine adopts the unusual syn glycosyl linkage, refuting the widespread belief that such conformations should all be anti. In contrast to the related d(TGGGGT)/daunomycin complex, there are no ligand–quadruplex groove insertion interactions.     

NMR solution structure of a highly stable de novo heterodimeric coiled-coil

The NMR solution structure of a highly stable coiled-coil IAAL-E3/K3 has been solved. The E3/K3 coiled-coil is a 42-residue de novo designed coiled-coil comprising three heptad repeats per subunit, stabilized by hydrophobic contacts within the core and electrostatic interactions at the interface crossing the hydrophobic core which direct heterodimer formation. This E3/K3 domain has previously been shown to have high alpha-helical content as well as possessing a low dissociation constant (70 nM). The E3/K3 structure is completely alpha-helical and is an archetypical coiled-coil in solution, as determined using a combination of (1)H-NOE and homology based structural restraints. This structure provides a structural framework for visualizing the important interactions for stability and specificity, which are key to protein engineering applications such as affinity purification and de novo design.     

NMR solution structure of the angiostatic peptide anginex

Anginex, a designed peptide 33mer, is known to function both as an antiangiogenic and bactericidal agent. Solving the NMR solution structure of the peptide is key to understand better its structure-activity relationships and to design more bioactive peptides and peptide mimetics. However, structure elucidation of anginex has been elusive due to subunit exchange-induced resonance broadening. Here, we found that performing NMR structural studies in a micellar environment abolishes exchange broadening and allows the structure of anginex to be determined. Anginex folds in an amphipathic, three-stranded antiparallel beta-sheet conformation with functionally key hydrophobic residues lying on one face of the beta-sheet and positively charged, mostly lysine residues, lying on the opposite face. Structural comparison is made with a homologous, yet relatively inactive peptide, betapep-28. These results contribute to the design of peptidomimetics of anginex for therapeutic use against angiogenically-related diseases like cancer, as well as infectious diseases.     

NMR solution structure of the neurotrypsin Kringle domain

Neurotrypsin is a multidomain protein that serves as a brain-specific serine protease. Here we report the NMR structure of its kringle domain, NT/K. The data analysis was performed with the BACUS (Bayesian analysis of coupled unassigned spins) algorithm. This study presents the first application of BACUS to the structure determination of a 13C unenriched protein for which no prior experimental 3D structure was available. NT/K adopts the kringle fold, consisting of an antiparallel beta-sheet bridged by an overlapping pair of disulfides. The structure reveals the presence of a surface-exposed left-handed polyproline II helix that is closely packed to the core beta-structure. This feature distinguishes NT/K from other members of the kringle fold and points toward a novel functional role for a kringle domain. Functional divergence among kringle domains is discussed on the basis of their surface and electrostatic characteristics.     

NMR solution structure of the non-RGD disintegrin obtustatin

The solution structure of obtustatin, a novel non-RGD disintegrin of 41 residues isolated from Vipera lebetina obtusa venom, and a potent and selective inhibitor of the adhesion of integrin alpha(1)beta(1) to collagen IV, has been determined by two-dimensional nuclear magnetic resonance. Almost the whole set of chemical shifts for 1H, 13C and 15N were assigned at natural abundance from 2D homonuclear and heteronuclear 500 MHz, 600 MHz and 800 MHz spectra at pH 3.0 recorded at 298 K and 303 K. Final structural constraints consisted of 302 non-redundant NOE (95 long-range, 60 medium, 91 sequential and 56 intra-residue), four disulfide bond distances, five chi1 dihedral angles and four hydrogen bonds. The 20 conformers with lowest total energy had no NOE violations greater than 0.35A or dihedral angle violations greater than 12 degrees. The average root-mean-square deviation (RMSD) for backbone atoms of all residues among the 20 conformers was 1.1A and 0.6A for the 29 best-defined residues. Obtustatin lacks any secondary structure. Compared to all known disintegrin structures in which the RGD motif is located at the apex of an 11 residue hairpin loop, the active KTS tripeptide of obtustatin is oriented towards a side of its nine residue integrin-binding loop. The C-terminal tail is near to the active loop, and these two structural elements display the largest atomic displacements due to local conformational disorder. Double cross-peaks for W20, Y28 and H27 in the aromatic region of TOCSY spectra, local RMSD values for these residues, and positive cross-peaks in a ROESY spectrum (600 MHz, 100 ms mixing time), suggest that these residues act as a hinge allowing for the overall flexibility of the entire integrin-binding loop. These distinct structural features, along with its different electrostatic surface potential in relation to other known disintegrins, may confer to obtustatin its reported alpha(1)beta(1) integrin inhibitory selectivity.