Abstract: The structure of recombinant domain III of the envelope protein (rED3) of yellow fever virus (YFV), containing the major neutralization site, was determined using NMR spectroscopy. The amino acid sequence and structure of the YFV-rED3 shows differences from ED3s of other mosquito-borne flaviviruses; in particular, the partially surface-exposed BC loop where methionine-304 and valine-324 were identified as being critical for the structure of the loop. Variations in the structure and surface chemistry of ED3 between flaviviruses affect neutralization sites and may affect host cell receptor interactions and play a role in the observed variations in viral pathogenesis and tissue tropism.
Abstract: The most abundant base-substitution mutation resulting from oxidative damage to DNA is the GC to AT transition mutation. 5-hydroxyuracil (5-OHU), produced by the oxidative deamination of cystosine, has been established as the major chemical precursor for this most abundant transition mutation. Results from NMR spectroscopy and UV melting experiments show that 5-OHU would form the most stable pair with G, and the least stable pair with C. The hydroxyl group in the 5th position of the 5-OHU residue may play a role in increasing the stability of the 5-OHU:G pair over the normal Watson-Crick pair, the 5-OHU:A. The 5-OHU:C base pair would be least stable, and would destabilize the base-stacking in the duplex. Our results explain why certain DNA polymerases preferentially incorporate G opposite to 5-OHU over A and why C does not get incorporated against 5-OHU during DNA replication in vivo.
Abstract: The disease dengue (DEN) is caused by four serologically related viruses termed DEN1, DEN2, DEN3 and DEN4. The structure of the ectodomain of the envelope protein has been determined previously for DEN2 and DEN3 viruses. Using NMR spectroscopic methods, we solved the solution structure of domain III (ED3), the receptor-binding domain, of the envelope protein of DEN4 virus, human strain 703-4. The structure shows that the nine amino acid changes in ED3 that separate the sylvatic and human DEN4 strains are surface exposed. Important structural differences between DEN4-rED3 and ED3 domains of DEN2, DEN3 and other flaviviruses are discussed.
Abstract: Oxidation of the thymine methyl group produces two stable products, non-mutagenic 5-hydroxymethyluracil and highly mutagenic 5-formyluracil. We have calculated the interaction energy of base-pair formation involving 5-formyluracil bound to the natural DNA bases adenine (A), cytosine (C), guanine (G), and thymine (T), and discuss the effects of the 5-formyl group with respect to similar base-pairs containing uracil, 5-hydroxyuracil, thymine (5-methyluracil), and 5-hydroxycytosine. The interaction geometries and energies were calculated four ways: (a) using density functional theory (DFT) without basis set super-position error (BSSE) corrections, (b) using DFT with BSSE correction of geometries and energies, (c) using Møller-Plesset second order perturbation theory (MP2) without BSSE correction, and (d) using MP2 with BSSE geometry and energy correction. All calculations used the 6-311G(d,p) basis set. Notably, we find that the A:5-formyluracil base-pair is more stable than the precursor A:T base-pair. The relative order of base-pair stabilities is A:5-Fo-U > G:5-Fo-U > C:5-Fo-U > T:5-Fo-U.
Abstract: Thioaptamers offer advantages over normal phosphate ester backbone aptamers due to their enhanced affinity, specificity, and higher stability, largely due to the properties of the sulfur backbone modifications. Over the past several years, in vitro thioaptamer selection and bead-based thioaptamer selection techniques have been developed in our laboratory. Furthermore, several thioaptamers targeting specific proteins such as transcription factor NF-kappaB and AP-1 proteins have been identified. Selected thioaptamers have been shown diagnostic promise in proteome screens. Moreover, some promising thioaptamers have been shown in preliminary animal therapeutic dosing to increase survival in animal models of infection with West Nile virus.
Abstract: VPgs are essential for replication of picornaviruses, which cause diseases such as poliomyelitis, foot and mouth disease, and the common cold. VPg in infected cells is covalently linked to the 5' end of the viral RNA, or, in a uridylylated form, free in the cytoplasm. We show here the first solution structure for a picornaviral VPg, that of the 22-residue peptide from poliovirus serotype 1. VPg in buffer is inherently flexible, but a single conformer was obtained by adding trimethylamine N-oxide (TMAO). TMAO had only minor effects on the TOCSY spectrum. However, it increased the amount of structured peptide, as indicated by more peaks in the NOESY spectrum and an up to 300% increase in the ratio of normalized NOE cross peak intensities to that in buffer. The data for VPg in TMAO yielded a well defined structure bundle with 0.6 A RMSD (versus 6.6 A in buffer alone), with 10-30 unambiguous constraints per residue. The structure consists of a large loop region from residues 1 to 14, from which the reactive tyrosinate projects outward, and a C-terminal helix from residues 18 to 21 that aligns the sidechains of conserved residues on one face. The structure has a stable docking position at an area on the poliovirus polymerase crystal structure identified as a VPg binding site by mutagenesis studies. Further, UTP and ATP dock in a base-specific manner to the reactive face of VPg, held in place by residues conserved in all picornavirus VPgs.
Abstract: We have solved the NMR solution structure of domain III from the Omsk hemorrhagic fever virus envelope protein and report the first sequencing of the Guriev strain of this virus. Important structural differences between tick-borne flaviviruses, such as OHFV and TBE, and mosquito-borne flaviviruses, such as West Nile virus, are discussed.
Abstract: The VPg peptide, which is found in poliovirus infected cells either covalently bound to the 5'-end of both plus and minus strand viral RNA, or in a uridylylated free form, is essential for picornavirus replication. Combining experimental structure and mutation results with molecular modeling suggests a new mechanism for VPg uridylylation, which assigns an additional function, that of scaffold, to the polymerase. The polarity of the NMR structure of VPg is complementary to the binding site on the surface of poliovirus polymerase determined previously by mutagenesis. Docking VPg at this position places the reactive tyrosinate close to the 5'-end of Poly(A)7 RNA when this is bound with its 3'-end in the active site of the polymerase. The triphosphate tail of a UTP moiety, base paired with the 5'-end of the RNA, projects back over the Tyr3-OH and is held in position by conserved positively charged side-chains of VPg. Other conserved residues mediate binding to the polymerase surface and serve as ligands for metal ion catalyzed transphosphorylation. Additional viral proteins or a second polymerase molecule may aid in stabilizing the components of the reaction. In the model complex, VPg can direct its own uridylylation before entering the polymerase active site.
Abstract: Oxidized cytosine product 5-hydroxyuracil has been shown to be the major chemical precursor for the GC to AT transition, the most frequent substitution mutation observed in aerobic organisms. We have calculated the interaction energy of base-pair formation involving uracil or 5-hydroxyuracil, which is formed in cells by oxidative deamination of cytosine, bound to any of the natural DNA bases, A, C, G, and T, and discuss the effects of the hydroxyl group in this respect. The base-pair geometries and energies were calculated using the 6-311G(dp) basis set under four conditions: using density functional theory (DFT) without out basis set super-position error (BSSE) correction, using DFT with BSSE correction of geometries and energies, using Møller-Plesset second order perturbation theory (MP2) without BSSE correction, and using MP2 with BSSE geometry and energy correction. We find that the hydroxyl group of 5-HO-U (relative to U) has little effect on the base-pairs with A, C or one conformation of T, while making a substantial energy difference in base-pairs involving G or a different conformation of T. For most of the complexes studied, the BSSE-corrected energies at the DFT and MP2 levels of theory agreed to within 0.5 kcal.
Abstract: NMR studies, UV-monitored melting experiments, and ab initio calculations show that 5-hydroxyuracil, produced by the oxidative de-amination of cytosines by reactive oxygen species, can form stable base-pairs with dA, dG, dC and dT residues in a DNA duplex, providing a basis for the in-vivo incorporation of 5-hydroxyuracil during DNA replication.
Abstract: The solution structure of domain III from the New York West Nile virus strain 385-99 (WN-rED3) has been determined by NMR methods. The West Nile domain III structure is a beta-barrel structure formed from seven anti-parallel beta-strands in two beta-sheets. One anti-parallel beta-sheet consists of beta-strands beta1 (Phe(299)-Asp(307)), beta2 (Val(313)-Tyr(319)), beta4 (Arg(354)-Leu(355)), and beta5 (Lys(370)-Glu(376)) arranged so that beta2 is flanked on either side by beta1 and beta5. The short beta4 flanks the end of the remaining side of beta5. The remaining anti-parallel beta-sheet is formed from strands beta3 (Ile(340)-Val(343)), beta6 (Gly(380)-Arg(388)), and beta7 (Gln(391)-Lys(399)) arranged with beta6 at the center. Residues implicated in antigenic differences between different West Nile virus strains (and other flaviviruses) and neutralization are located on the outer surface of the protein. Characterization of the binding of monoclonal antibodies to WN-rED3 mutants, which were identified through neutralization escape experiments, indicate that antibody neutralization directly correlates with binding affinities. These studies provide an insight into theoretical virus-receptor interaction points, structure of immunogenic determinants, and potential targets for antiviral agents against West Nile virus and highlight differences between West Nile virus and other flavivirus structures that may represent critical determinants of virulence.
Abstract: A number of transcription factor proteins contain domains that are fully or partially unstructured. The means by which such proteins acquire naturally folded conformations are not well understood. When they encounter their proper binding partner(s), several of these proteins adopt a folded conformation through an induced-fit mechanism. The glucocorticoid receptor (GR) is a ligand-activated transcription factor. Expressed independently as a recombinant peptide, the N-terminal transactivation domain (AF1) of the GR shows little structure and appears to exist as a collection of random coil configurations. The GR AF1 is known to interact with other transcription factors, including a critical component of the general transcription machinery proteins, the TATA box binding protein (TBP). We tested whether this interaction can lead to acquisition of structure in the GR AF1. Our results show that recombinant GR AF1 acquires a significant amount of helical content when it interacts with TBP. These structural changes were monitored by Fourier transform infrared and NMR spectroscopies, and by proteolytic digestions. Our results support a model in which TBP binding interaction with the GR AF1 induces significantly greater helical structure in the AF1 domain. This increased helical content in the GR AF1 appears to come mostly at the expense of random coil conformation. These results are in accordance with the hypothesis that an induced-fit mechanism gives structure to the GR AF1 when it encounters TBP.
Abstract: The efficiency of DNA glycosylases to initiate base excision repair (BER) has been demonstrated to be modulated by the precise sequence context in which the lesion or mismatch is located. In the case of DNA containing an A/G mismatch, in which the recognition and excision of adenine from the mismatch is mediated by the Escherichia coli MutY enzyme, not only does the local sequence context affect the strength of base stacking interactions, but it also modulates the syn/anti conformation around the glycosyl bond of the bases in the mispair. Utilizing prior NMR data to identify DNA sequence contexts that adopt either an anti/anti or a syn/anti configuration at an A/G mismatch, we tested the hypothesis that the initial equilibrium of the mismatched base orientations would modulate the overall efficiency of glycosyl bond scission. By systematically varying the sequence context around a central A/G mismatch within a 30-mer duplex DNA, significant kinetic differences were observed that were consistent with this hypothesis. Since the relative efficiency of the kinetics fell into only two groupings, a NMR study was conducted on a DNA sequence context of unknown syn/anti conformation. These data established that the relative syn/anti conformation did not correlate with the excision efficiency, as well as there being a lack of correlation between kinetics and thermal stability of these DNAs.
Abstract: The solution structure of an 11-mer DNA duplex, d(CGGTCA*CGAGG) x d(CCTCGTGACCG), containing a 10R adduct at dA* that corresponds to the cis addition of the N(6)-amino group of dA(6) to (+)-(9S,10R)-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied by 2D NMR methods. The NOESY cross-peak patterns indicate that the hydrocarbon is intercalated on the 5'-side of the modified base. This observation is the same as that observed for other oligonucleotides containing (10R)-dA adducts but opposite to that observed for the corresponding (10S)-dA adducts which are intercalated on the 3'-side of the modified base. The hydrocarbon is intercalated from the major groove without significant disruption of either the anti glycosidic torsion angle of the modified residue or the base pairing of the modified residue with the complementary residue on the opposite strand. The ensemble of 10 structures determined exhibits relatively small variations (6-15 degrees) in the characteristic hydrocarbon-base dihedral angles (alpha' and beta') as well as the glycosidic torsion angle chi. These angles are similar to those in a previously determined cis-opened benzo[a]pyrene diol epoxide-(10R)-dA adduct structure. Comparison of the present structure with the cis-opened diol epoxide adduct suggests that the absence of the 7- and 8-hydroxyl groups results in more efficient stacking of the aromatic moiety with the flanking base pairs and deeper insertion of the hydrocarbon into the helix. Relative to normal B-DNA, the duplex containing the present tetrahydroepoxide adduct is unwound at the lesion site, whereas the diol epoxide adduct structure is more tightly wound than normal B-DNA. Buckling of the adducted base pair as well as the C(5)-G(18) base pair that lies immediately above the hydrocarbon is much less severe in the present adducted structure than its cis-opened diol epoxide counterpart.
Abstract: Although the high-resolution structure of a protein may provide significant insight into which regions are important for function, it is well-known that proteins undergo significant conformational fluctuations, even under native conditions. This suggests that the static structure alone may not provide sufficient information for elucidation of the thermodynamic determinants of biological function and that an accurate molecular-level description of function requires knowledge of the nature and energetics of the conformational states that constitute the native state ensemble. Here the native state ensemble of the C-terminal src homology domain-3 (C-SH3) from Caenorhabditis elegans Sem-5 has been studied using a variety of high-resolution biophysical techniques. In addition to determining the first solution structure of the unliganded protein, we have performed (15)N relaxation and native state hydrogen-deuterium exchange. It is observed that the regions of greatest structural variabilility also show low protection and order parameters, suggesting a higher degree of conformational diversity. These flexible regions also coincide with those regions of Sem-5 that have been predicted by the COREX algorithm to be unfolded in many of the most probable conformational states within the native state ensemble. The implications of this agreement and the potential role of conformational heterogeneity of the observed biophysical properties are discussed.
Abstract: A variety of monothio- and dithiosubstituted duplex aptamers targeting NF-kappaB have been synthesized and designed. The specificity and affinity of the dithioate aptamers of p50 and RelA(p65) NF-kappaB homodimers was determined by gel shift experiments. The NMR solution structures for several unmodified and dithioate backbone modified 14-base paired duplex aptamers have been determined by a hybrid, complete matrix (MORASS)/restrained molecular dynamics method. Structural perturbations of the dithioate substitutions support our hypothesis that the dithioate binds cations less tightly than phosphoryl groups. This increases the electrostatic repulsion across the B-form narrow minor groove and enlarges the minor groove, similar to that found in A-form duplexes. Structural analysis of modeled aptamer complexes with NF-kappaB homo- and heterodimers suggests that the dithioate backbone substitution can increase the aptamer's relative affinity to basic groups in proteins such as NF-kappaB by helping to "strip" the cations from the aptamer backbone.
Abstract: To understand the structural basis of the recognition and removal of specific mismatched bases in double-stranded DNAs by the DNA repair glycosylase MutY, a series of structural and functional analyses have been conducted. MutY is a 39-kDa enzyme from Escherichia coli, which to date has been refractory to structural determination in its native, intact conformation. However, following limited proteolytic digestion, it was revealed that the MutY protein is composed of two modules, a 26-kDa domain that retains essential catalytic function (designated p26MutY) and a 13-kDa domain that is implicated in substrate specificity and catalytic efficiency. Several structures of the 26-kDa domain have been solved by X-ray crystallographic methods to a resolution of up to 1.2 A. The structure of a catalytically incompetent mutant of p26MutY complexed with an adenine in the substrate-binding pocket allowed us to propose a catalytic mechanism for MutY. Since reporting the structure of p26MutY, significant progress has been made in solving the solution structure of the noncatalytic C-terminal 13-kDa domain of MutY by NMR spectroscopy. The topology and secondary structure of this domain are very similar to that of MutT, a pyrophosphohydrolase. Molecular modeling techniques employed to integrate the two domains of MutY with DNA suggest that MutY can wrap around the DNA and initiate catalysis by potentially flipping adenine and 8-oxoguanine out of the DNA helix.
Abstract: One of the functions of MutY from Escherchia coli is removal of adenine mispaired with 7,8-dihydro-8-oxoguanine (8-oxoG), a common lesion in oxidatively damaged DNA. MutY is composed of two domains: the larger N-terminal domain (p26) contains the catalytic properties of the enzyme while the C-terminal domain (p13) affects substrate recognition and enzyme turnover. On the basis of sequence analyses, it has been recently suggested that the C-terminal domain is distantly related to MutT, a dNTPase which hydrolyzes 8-oxo-dGTP [Noll et al. (1999) Biochemistry 38, 6374-6379]. We have studied the solution structure of the C-terminal domain of MutY by NMR and find striking similarity with the reported solution structure of MutT. Despite low sequence identity between the two proteins, they have similar secondary structure and topology. The C-terminal domain of MutY is composed of two alpha-helices and five beta-strands. The NOESY data indicate that the protein has two beta-sheets. MutT is also a mixed alpha/beta protein with two helices and two beta-sheets composed of five strands. The secondary structure elements are similarly arranged in the two proteins.
Abstract: 2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.
