Max A. Keniry

Quadruplex structures in nucleic acids

DNA oligonucleotides that have repetitive tracts of guanine bases can form G‐quadruplex structures that display an amazing polymorphism. Structures of several new G‐quadruplexes have been solved recently that greatly expand the known structural motifs observed in nucleic acid quadruplexes. Base triads, base hexads, and quartets that contain cytosine have recently been identified stacked over the familiar G‐quartets. The current status of the diverse array of structural features in quadruplexes is described and used to provide insight into the polymorphism and folding pathways. This review also summarizes recent progress in the techniques used to probe the structures of G‐quadruplexes and discusses the role of ion binding in quadruplex formation. Several of the quadruplex structures featured in this review can be accessed in the online version of this review as CHIME representations.

NMR Structure Refinement and Dynamics of the K+-[d(G3T4G3)]2 Quadruplex via Particle Mesh Ewald Molecular Dynamics Simulations

The solution structure and dynamical properties of the potassium-stabilized, hairpin dimer quadruplex formed by the oligonucleotide d(G3T4G3) have been elucidated by a combination of high-resolution NMR and molecular dynamics simulations. Refinement calculations were carried out both in vacuo, without internally coordinated K+ cations, and in explicit water, with internally coordinated K+ cations. In the latter case, the electrostatic interactions were calculated using the particle mesh Ewald (PME) method. The NMR restraints indicate that the K+ quadruplex has a folding arrangement similar to that formed by the same oligonucleotide in the presence of sodium, but with significant local differences. Unlike the Na+ quadruplex, the thymine loops found in K+ exhibit considerable flexibility, and appear to interconvert between two preferred conformations. Furthermore, the NMR evidence points toward K+-stabilized guanine quartets of slightly larger diameter relative to the Na+-stabilized structure. The characteristics of the quartet stem are greatly affected by the modeling technique employed: caged cations alter the size and symmetry of the quartets, and explicit water molecules form hydration spines within the grooves. These results provide insight into those factors that determine the overall stability of hairpin dimer quadruplexes and the effects of different cations in modulating the relative stability of the dimeric hairpin and linear, four-stranded, quadruplex forms.

Solution structure of Domains IVa and V of the τ subunit of Escherichia coli DNA polymerase III and interaction with the α subunit

The solution structure of the C-terminal Domain V of the τ subunit of E. coli DNA polymerase III was determined by nuclear magnetic resonance (NMR) spectroscopy. The fold is unique to τ subunits. Amino acid sequence conservation is pronounced for hydrophobic residues that form the structural core of the protein, indicating that the fold is representative for τ subunits from a wide range of different bacteria. The interaction between the polymerase subunits τ and α was studied by NMR experiments where α was incubated with full-length C-terminal domain (τC16), and domains shortened at the C-terminus by 11 and 18 residues, respectively. The only interacting residues were found in the C-terminal 30-residue segment of τ, most of which is structurally disordered in free τC16. Since the N- and C-termini of the structured core of τC16 are located close to each other, this limits the possible distance between α and the pentameric δτ2γδ′ clamp–loader complex and, hence, between the two α subunits involved in leading- and lagging-strand DNA synthesis. Analysis of an N-terminally extended construct (τC22) showed that τC14 presents the only part of Domains IVa and V of τ which comprises a globular fold in the absence of other interaction partners.

Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit.

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, epsilon, and theta subunits. The theta subunit is the smallest and least understood subunit. The three-dimensional structure of theta in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of epsilon186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of theta in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed theta show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free theta (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of theta in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon186 on theta was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of theta are located about 15 A or farther from the lanthanide ion in the active site of epsilon186, in agreement with the extensive biochemical data for the theta-epsilon system.

A comparison of the association of spermine with duplex and quadruplex DNA by NMR

The association of [1′,1″‐13C2]spermine ([1′,1″‐13C2]N,N′‐bis(3‐aminopropyl)‐1,4‐butanediamine) with duplex and quadruplex DNA has been studied by nuclear magnetic resonance spectroscopy. 1D NOESY experiments using two‐way selective cross‐polarization (ISI‐SCP‐NOESY) showed spermine intramolecular NOEs are either weakly positive or weakly negative when spermine is complexed to duplex B‐DNA and linear four‐stranded quadruplex DNA. In contrast, large negative intramolecular NOEs are observed when spermine is complexed to two distinct forms of folded quadruplex DNA suggesting greater immobilization of spermine on these folded DNA quadruplexes. No changes in the quadruplex stem structure are observed but there are minor changes to the loop structure of a two‐stranded folded quadruplex on binding spermine.

The effect of gossypol and 6-aminonicotinamide on tumor cell metabolism: a 31P-magnetic resonance spectroscopic study

31P-Magnetic resonance spectroscopy has been used to assess the changes in the levels of water-soluble phosphate pools in T47-D breast carcinoma cells induced by the antimitochondrial drugs, gossypol and 6-aminonicotinamide. A decrease in the NTP/Pi ratio occurred after treatment with gossypol. No change in the NTP/Pi ratio occurred on treatment with 6-aminonicotinamide; however, a substantial accumulation of 6-phosphogluconate was observed. Pretreatment of T47-D cells with gossypol prevented the accumulation of 6-phosphogluconate. This facile and non-invasive approach suggests that the oxidative part of the pentose-phosphate shuttle is an important source of reducing equivalents in T47-D cells. This pathway may prove to be a useful target for site-directed drug attack in carcinoma cell lines that require large quantities of NADP for the synthesis of fatty acids and steroids.

The three-dimensional structure of the 4:1 mithramycin:d(ACCCGGGT)2 complex: evidence for an interaction between the E saccharides

Mithramycin and chromomycin, two antitumor drugs, each having an identical aglycone and nearly identical disaccharide and trisaccharide side chains, have differing binding properties to a small oligonucleotide, d(ACCCGGGT)(2) (M. A. Keniry et al., Journal of Molecular Biology, 1993, Vol. 231, pp. 753-767). In order to understand the forces that induce four mithramycin molecules to bind to d(ACCCGGGT)(2) instead of two drug molecules in the case of chromomycin, the structure of the 4:2:1 mithramycin: Mg(2+):d(ACCCGGGT)(2) complex was investigated by (1)H-nmr and restrained molecular dynamics. The resulting three-dimensional model showed that in order to accommodate the close approach of one neighboring mithramycin dimer, the inwardly directed CDE saccharide chain of the neighboring mithramycin dimer undergoes a conformational change such that the E saccharide no longer spans the minor groove but reorients so that the hydrophilic face of the E saccharides from the two dimers oppose each other. Two hydrogen bonds are formed between the hydroxyl groups of the two opposing E saccharide groups. The results are interpreted in terms of the differences in stereochemistry and functional group substitutions between mithramycin and chromomycin. A mithramycin dimer is able to self-associate on an oligonucleotide template because it has two hydroxyl groups on the same face of its terminal E saccharide. A chromomycin dimer is unable to self-associate because one of these hydroxyl groups is acetylated and the neighboring hydroxyl group has a stereochemistry that cannot permit close contact of the hydroxyl group with a neighbouring chromomycin dimer.

[24] NMR studies of drug-DNA complexes

Publisher Summary This chapter discusses the practical aspects of carrying out high-resolution nuclear magnetic resonance (NMR) experiments to investigate drug-DNA complexes. In addition to the large number of recent studies based on the NMR analysis of the solution structure of the drug-DNA complexes, there have been a substantial number of structural studies based on X-ray crystallographic analysis of such complexes. Once crystals have been obtained that diffract well, this approach has the advantage that high-resolution structures can often be determined in a more direct fashion compared to the NMR approach. Several important differences remain, however, between structure determination in solution and in the solid state. First, in case of nucleic acids, their conformation is frequently different in the solid and solution state, because of hydration and/or crystal packing effects. Second, it is considerably easier to explore the effects of changing temperature and salt conditions, in solution to learn something, about the kinetics of interaction, between the drug and the DNA. Third, studies to examine the stoichiometry of various drug-DNA complexes are more easily carried out in solution than in the solid state. For these reasons, much can be learned from the structural analysis carried out in solution that is complementary to the information available from the X-ray crystallographic studies.

Insight into the molecular recognition of spermine by DNA quadruplexes from an NMR study of the association of spermine with the thrombin-binding aptamer

The preferred residence sites and the conformation of DNA‐bound polyamines are central to understanding the regulatory roles of polyamines. To this end, we have used a series of selective 13C‐edited and selective total correlation spectroscopy‐edited one‐dimensional (1D) nuclear Overhauser effect spectroscopy NMR experiments to determine a number of intramolecular 1H nuclear Overhauser effect (NOE) connectivities in 13C‐labelled spermine bound to the thrombin‐binding aptamer. The results provide evidence that the aptamer‐bound spermine adopts a conformation that optimizes electrostatic and hydrogen bond contacts with the aptamer backbone. The distance between the nitrogen atoms of the central aminobutyl is reduced by an increase in the population of gauche conformers at the C6–C7 bonds, which results in either a curved or S‐shaped spermine conformation. Molecular modelling contributes insight toward the mode of spermine binding of these spermine structures within the narrow grooves of DNA quadruplexes. In each case, the N5 ammonium group makes hydrogen bonds with two nearby phosphates across the narrow groove. Our results have implications for the understanding of chromatin structure and the rational design of quadruplex‐binding drugs. Copyright

Secondary structure determination of 15N-labelled human Long-[Arg-3]-insulin-like growth factor 1 by multidimensional NMR spectroscopy

Insulin‐like growth factors (IGFs) are a group of proteins that promote cell growth and differentiation. Long‐[Arg‐3]‐IGF‐I (Francis et al. (1992) J. Mol. Endocrinol. 8, 213–223), a potent analogue of IGF‐I, which has a Glu‐3 to Arg‐3 substitution and a hydrophobic, thirteen amino acid N‐terminal extension, has been studied by 1H,15N NMR spectroscopy. All the backbone 1H and 15N assignments and most of the 1H sidechain assignments have been completed. The secondary structure elements were identified by determining the sequential and medium range NOEs from sensitivity‐enhanced 15N‐NOESY‐HSQC and sensitivity‐enhanced 15N‐HSQC‐NOESY‐HSQC spectra. The IGF‐I domain of Long‐[Arg‐3]‐IGF‐I was found to have an almost identical structure to IGF‐I. The N‐terminal seven amino acid residues of the extension have very few medium range or long range NOEs but the next five amino acids form a turn‐like structure that is spatially close to the beginning of helix 1 in the IGF‐I domain. Hydrogen‐deuterium exchange experiments show that all the slowly exchanging backbone amide protons in the IGF‐I domain are either in the helical or the extended structural elements. Many of the amide protons in the N‐terminal extension are also protected from the solvent although the residues in this part of the extension do not have any identifiable secondary structure. The results are interpreted in terms of the increased biological potency of Long‐[Arg‐3]‐IGF‐I and the decreased binding to insulin‐like growth factor binding proteins.