Calculations of binding affinity between C8-substituted GTP analogs and the bacterial cell-division protein FtsZ

The FtsZ protein is a self-polymerizing GTPase that plays a central role in bacterial cell division. Several C8-substituted GTP analogs are known to inhibit the polymerization of FtsZ by competing for the same binding site as its endogenous activating ligand GTP. Free energy calculations of the relative binding affinities to FtsZ for a set of five C8-substituted GTP analogs were performed. The calculated values agree well with the available experimental data, and the main contribution to the free energy differences is determined to be the conformational restriction of the ligands. The dihedral angle distributions around the glycosidic bond of these compounds in water are known to vary considerably depending on the physicochemical properties of the substituent at C8. However, within the FtsZ protein, this substitution has a negligible influence on the dihedral angle distributions, which fall within the narrow range of −140° to −90° for all investigated compounds. The corresponding ensemble average of the coupling constants 3J(C4,H1′) is calculated to be 2.95 ± 0.1 Hz. The contribution of the conformational selection of the GTP analogs upon binding was quantified from the corresponding populations. The obtained restraining free energy values follow the same trend as the relative binding affinities to FtsZ, indicating their dominant contribution

Guanine bases in DNA G-quadruplex adopt nonplanar geometries owing to solvation and base pairing

The effect of base pairing and solvation on pyramidalization of the glycosidic nitrogen found in the residues of parallel G-quadruplex with NDB ID UDF062 is analyzed and explained with theoretical calculations. The extent of the pyramidalization depends on the local geometry of the 2′-deoxyguanosine residues, namely on their glycosidic torsion and sugar pucker, which are predetermined by the 3D-architecture of G-quadruplex. Pyramidal inversion of the glycosidic nitrogen found in 2′-deoxyguanosines of G-quadruplex is induced owing to site-specifically coordinated solvent. Different adiabatic structural constraints used for fixing the base-to-sugar orientation of 2′-deoxyguanosine in geometry optimizations result in different extents of pyramidalization and induce pyramidal inversion of the glycosidic nitrogen. These model geometry constraints helped us analyze the effect of real constraints represented by explicit molecular environment of selected residues of the G-quadruplex. The maximal extent of the glycosidic nitrogen pyramidalization found in the high-resolution crystal structure corresponds to the calculation to deformation energy of only 1 kcal mol–1. The out-of-plane deformations of nucleobases thus provide a way for compensating the site-specific external environmental stress on the G-quadruplex

Evaluating the effects of the nonplanarity of nucleic acid bases on NMR, IR, and vibrational circular dichroism spectra: a density functional theory computational study

The pyramidalizations of N9/1 glycosidic nitrogens in DNA and RNA nucleosides, recently discovered and analyzed in their ultrahigh-resolution X-ray crystal structures (Sychrovsky´; et al. Nucleic Acid Res. 2009, 37, 7321.), were found to have significant effects on the structural interpretation of the 3J(C4/2-H1′) and 3J(C8/ 6-H1′) NMR scalar couplings in purine/pyrimidine nucleosides. The calculated effects on IR and vibrational circular dichroism (VCD) spectra were only minor. The calculated structural deformations in nucleosides, depending on sugar-to-base orientation, gave rise to corrections in the phase shift of the Karplus equations for the 3J(C8/6-H1′) and 3J(C4/2-H1′) couplings ranging from -26° to +25° and from -5.7° to +2.0°, respectively. The sign alternation of this correction in syn and anti nucleosides arises from the stereoinversion of the N9/1 glycosidic nitrogen occurring upon reorientation of the glycosidic torsion. The effect was calculated consistently in the dG, dA, dC, dT, rA, and rG nucleosides. Utilization of the calculated phase-shift corrections in the design of Karplus equations for the 3J couplings was suggested, and the effects on structural interpretation of the experimental couplings were evaluated

Guanine bases in DNA G-quadruplex adopt nonplanar geometries owing to solvation and base pairing

The effect of base pairing and solvation on pyramidalization of the glycosidic nitrogen found in the residues of parallel G-quadruplex with NDB ID UDF062 is analyzed and explained with theoretical calculations. The extent of the pyramidalization depends on the local geometry of the 2′-deoxyguanosine residues, namely on their glycosidic torsion and sugar pucker, which are predetermined by the 3D-architecture of G-quadruplex. Pyramidal inversion of the glycosidic nitrogen found in 2′-deoxyguanosines of G-quadruplex is induced owing to site-specifically coordinated solvent. Different adiabatic structural constraints used for fixing the base-to-sugar orientation of 2′-deoxyguanosine in geometry optimizations result in different extents of pyramidalization and induce pyramidal inversion of the glycosidic nitrogen. These model geometry constraints helped us analyze the effect of real constraints represented by explicit molecular environment of selected residues of the G-quadruplex. The maximal extent of the glycosidic nitrogen pyramidalization found in the high-resolution crystal structure corresponds to the calculation to deformation energy of only 1 kcal mol–1. The out-of-plane deformations of nucleobases thus provide a way for compensating the site-specific external environmental stress on the G-quadruplex

Investigation of Quadruplex Structure Under Physiological Conditions Using In-Cell NMR

In this chapter we describe the application of in-cell NMR spectroscopy to the investigation of G-quadruplex structures inside living Xenopus laevis oocytes and in X. laevis egg extract. First, in-cell NMR spectroscopy of nucleic acids (NA) is introduced and applications and limitations of the approach are discussed. In the following text the application of in-cell NMR spectroscopy to investigation of G-quadruplexes are reviewed. Special emphasis is given to the discussion of the influence of the intracellular environmental factors such as low molecular weight compounds, molecular crowding, and hydration on structural behavior of G-quadruplexes. Finally, future perspectives of in-cell NMR spectroscopy for quantitative characterization of G-quadruplexes and NA are discussed

Pyramidalization of the Glycosidic Nitrogen Provides the Way for Efficient Cleavage of the N‑Glycosidic Bond of 8‑OxoG with the hOGG1 DNA Repair Protein

A mechanistic pathway for cleavage of the N-glycosidic bond of 8-oxo-2′-deoxyguanosine (oxoG) catalyzed with the human 8-oxoguanine glycosylase 1 DNA repair protein (hOGG1) is proposed in this theoretical study. The reaction scheme suggests direct proton addition to the glycosidic nitrogen N9 of oxoG from the Nε-ammonium of Lys249 residue of hOGG1 that is enabled owing to the N9 pyramidal geometry. The N9-pyramidalization of oxoG is induced within hOGG1 active site. The coordination of N9 nitrogen to the Nε-ammonium of Lys249 unveiled by available crystal structures enables concerted, synchronous substitution of the N9−C1′ bond by the N9−H bond. The reaction is compared with other pathways already proposed by means of calculated activation energies. The ΔG# energy for the newly proposed reaction mechanism calculated with the B3LYP/6-31G(d,p) method 17.0 kcal mol−1 is significantly lower than ΔG# energies for other reactions employing attack of the Nε-amino group to the anomeric carbon C1′ of oxoG and attack of the Nε-ammonium to the N3 nitrogen of oxoG base. Moreover, activation energy for the oxoG cleavage proceeding via N9-pyramidalization is lower than energy calculated for normal G because the electronic state of the five-membered aromatic ring of oxoG is better suited for the reaction. The modification of aromatic character introduced by oxidation to the nucleobase thus seems to be the factor that is checked by hOGG1 to achieve base-specific cleavage

Fluorine Labeling as a Versatile Tool for Probing Nucleic Acid Folding and Interactions by NMR Spectroscopy

This mini-review provides an overview of approaches to19F-labeling of nucleic acids. A special attention is paid to applications of19F-labeled nucleic acids to resolve their polymorphism and characterize their folding and interactions with ligands and proteins in vitro and in living cells by using NMR spectroscopy

Solution structures of stem–loop RNAs that bind to the two N-terminal RNA-binding domains of nucleolin

Nucleolin, a multi-domain protein involved in ribosome biogenesis, has been shown to bind the consensus sequence (U/G)CCCG(A/G) in the context of a hairpin loop structure (nucleolin recognition element; NRE). Previous studies have shown that the first two RNA-binding domains in nucleolin (RBD12) are responsible for the interaction with the in vitro selected NRE (sNRE). We have previously reported the structures of nucleolin RBD12, sNRE and nucleolin RBD12–sNRE complex. A comparison of free and bound sNRE shows that the NRE loop becomes structured upon binding. From this observation, we hypothesized that the disordered hairpin loop of sNRE facilitates conformational rearrangements when the protein binds. Here, we show that nucleolin RBD12 is also sufficient for sequence- specific binding of two NRE sequences found in pre-rRNA, b1NRE and b2NRE. Structural investigations of the free NREs using NMR spectroscopy show that the b1NRE loop is conformationally heterogeneous, while the b2NRE loop is structured. The b2NRE forms a hairpin capped by a YNMG-like tetraloop. Comparison of the chemical shifts of sNRE and b2NRE in complex with nucleolin RBD12 suggests that the NRE consensus nucleotides adopt a similar conformation. These results show that a disordered NRE consensus sequence is not a prerequisite for nucleolin RBD12 binding

Investigation of Quadruplex Structure Under Physiological Conditions Using In-Cell NMR

In this chapter we describe the application of in-cell NMR spectroscopy to the investigation of G-quadruplex structures inside living Xenopus laevis oocytes and in X. laevis egg extract. First, in-cell NMR spectroscopy of nucleic acids (NA) is introduced and applications and limitations of the approach are discussed. In the following text the application of in-cell NMR spectroscopy to investigation of G-quadruplexes are reviewed. Special emphasis is given to the discussion of the influence of the intracellular environmental factors such as low molecular weight compounds, molecular crowding, and hydration on structural behavior of G-quadruplexes. Finally, future perspectives of in-cell NMR spectroscopy for quantitative characterization of G-quadruplexes and NA are discussed