NMR determination of the hetero-association of phenanthridines with daunomycin and their competitive binding to a DNA oligomer

500 MHz (1)H NMR spectroscopy has been used to determine thermodynamic and structural information on the hetero-association of daunomycin (DAU) with the phenanthridine mutagenic dyes ethidium bromide (EB) and propidium iodide (PI). The NMR complexation data have been analysed by a statistical-thermodynamic model which takes into account indefinite association for both the self-association of the drugs and their hetero-association. The results have been used to estimate the effect of the side chains of the phenanthridines on the competitive binding between DAU and the mutagens with DNA. Knowledge of the equilibrium constants for self-association of the phenanthridines and DAU, their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the EB-DAU, PI-DAU, EB-DAU-d(TGCA) and PI-DAU-d(TGCA) complexes to be calculated as a function of drug concentration in mixed solutions. The results provide some insight into the molecular basis of the action of combinations of biologically-active molecules. When intercalating drugs are used in combination, it is found that the decrease in binding of drug or mutagen with DNA is due both to formation of drug-mutagen hetero-association complexes in the mixed solution and to competition for the binding sites by the aromatic molecules; the relative importance of each process depends on the molecular properties of the drug or mutagen molecules being considered. Thus, the longer branched side chain of PI and the electrostatic contribution of the extra positive charge of the molecule compared with the ethyl group of EB results in lower affinity for self-association of PI molecules and their hetero-association with DAU, but increases the degree of binding of PI with DNA.     

Complexation of norfloxacin with DNA in the presence of caffeine

(1)H NMR spectroscopy (500 MHz) has been used to quantify the complexation of the antibacterial antibiotic Norfloxacin (NOR) with DNA in the presence of Caffeine (CAF). Separate studies have been made for the self-association of NOR, its hetero-association with CAF and complexation with a model self-complementary DNA tetramer, 5'-d(TpGpCpA), in order to determine the equilibrium parameters (induced chemical shifts, association constants, enthalpy and entropy) of the two-component mixtures to aid the analysis of the three-component systems. Investigations of the self-association of NOR and its hetero-association with CAF show that the aggregation of NOR molecules and association with CAF in solution are driven by the stacking of aromatic chromophores. The complexation of NOR with d(TGCA) has been analysed in terms of intercalation with the double-stranded form and non-intercalative binding with the single-stranded form of DNA. Investigations of the competitive binding of NOR and CAF with DNA show that at physiological concentrations of NOR (muM) and CAF (mM) the dominant mechanism influencing the affinity of NOR with DNA is the displacement of bound NOR molecules from DNA due to CAF-DNA complexation (i.e. the protector action of Caffeine).     

Area per lipid and acyl length distributions in fluid phosphatidylcholines determined by (2)H NMR spectroscopy

Deuterium ((2)H) NMR spectroscopy provides detailed information regarding the structural fluctuations of lipid bilayers, including both the equilibrium properties and dynamics. Experimental (2)H NMR measurements for the homologous series of 1, 2-diacyl-sn-glycero-3-phosphocholines with perdeuterated saturated chains (from C12:0 to C18:0) have been performed on randomly oriented, fully hydrated multilamellar samples. For each lipid, the C-D bond order parameters have been calculated from de-Paked (2)H NMR spectra as a function of temperature. The experimental order parameters were analyzed using a mean-torque potential model for the acyl chain segment distributions, and comparison was made with the conventional diamond lattice approach. Statistical mechanical principles were used to relate the measured order parameters to the lipid bilayer structural parameters: the hydrocarbon thickness and the mean interfacial area per lipid. At fixed temperature, the area decreases with increasing acyl length, indicating increased van der Waals attraction for longer lipid chains. However, the main effect of increasing the acyl chain length is on the hydrocarbon thickness rather than on the area per lipid. Expansion coefficients of the structural parameters are reported and interpreted using an empirical free energy function that describes the force balance in fluid bilayers. At the same absolute temperature, the phosphatidylcholine (PC) series exhibits a universal chain packing profile that differs from that of phosphatidylethanolamines (PE). Hence, the lateral packing of phospholipids is more sensitive to the headgroup methylation than to the acyl chain length. A fit to the area per lipid for the PC series using the empirical free energy function shows that the PE area represents a limiting value for the packing of fluid acyl chains.     

Location of the Zn(2+)-binding site on S100B as determined by NMR spectroscopy and site-directed mutagenesis

In addition to binding Ca(2+), the S100 protein S100B binds Zn(2+) with relatively high affinity as confirmed using isothermal titration calorimetry (ITC; K(d) = 94 +/- 17 nM). The Zn(2+)-binding site on Ca(2+)-bound S100B was examined further using NMR spectroscopy and site-directed mutagenesis. Specifically, ITC measurements of S100B mutants (helix 1, H15A and H25A; helix 4, C84A, H85A, and H90A) were found to bind Zn(2+) with lower affinity than wild-type S100B (from 2- to >25-fold). Thus, His-15, His-25, Cys-84, His-85, and perhaps His-90 of S100B are involved in coordinating Zn(2+), which was confirmed by NMR spectroscopy. Previous studies indicate that the binding of Zn(2+) enhances calcium and target protein-binding affinities, which may contribute to its biological function. Thus, chemical shift perturbations observed here for residues in both EF-hand domains of S100B during Zn(2+) titrations could be detecting structural changes in the Ca(2+)-binding domains of S100B that are pertinent to its increase in Ca(2+)-binding affinity in the presence of Zn(2+). Furthermore, Zn(2+) binding causes helix 4 to extend by one full turn when compared to Ca(2+)-bound S100B. This change in secondary structure likely contributes to the increased binding affinity that S100B has for target peptides (i.e., TRTK peptide) in the presence of Zn(2+).     

Interaction of a goose-type lysozyme with chitin oligosaccharides as determined by NMR spectroscopy

The interaction between a goose-type lysozyme from ostrich egg white (OEL) and chitin oligosaccharides [(GlcNAc)(n) (n = 2, 4 and 6)] was studied by nuclear magnetic resonance (NMR) spectroscopy. A stable isotope-labelled OEL was produced in Pichia pastoris, and backbone resonance assignments for the wild-type and an inactive mutant (E73A OEL) were achieved using modern multi-dimensional NMR techniques. NMR titration was performed with (GlcNAc)(n) for mapping the interaction sites of the individual oligosaccharides based on the shifts in the two-dimensional heteronuclear single quantum correlation (HSQC) resonances. In wild-type OEL, the interaction sites for (GlcNAc)(n) were basically similar to those determined by X-ray crystallography. In E73A OEL, however, the interaction sites were spread more widely over the substrate-binding cleft than expected, due to the multiple modes of binding. The association constant for E73A OEL and (GlcNAc)(6) calculated from the shifts in the Asp97 resonance (7.2 × 10(3) M(-1)) was comparable with that obtained by isothermal titration calorimetry (5.3 × 10(3) M(-1)). The interaction was enthalpy-driven as judged from the thermodynamic parameters (ΔH = -6.1 kcal/mol and TΔS = -1.0 kcal/mol). This study provided novel insights into the oligosaccharide binding mechanism and the catalytic residues of the enzymes belonging to family GH-23.     

Complexation of anthracycline drugs with DNA in the presence of caffeine

The competitive binding of anthracycline antitumour drugs, [daunomycin (DAU), doxorubicin (DOX) or nogalamycin (NOG)], with caffeine (CAF) to a model DNA oligomer has been investigated by 500 MHz ¹H NMR spectroscopy under physiological solution conditions. The method depends on the stepwise analysis of one-component (self-association), two-component (hetero-association and DNA complexation) and three-component interactions, in order to de-convolute the overall binding of the anthracycline antibiotic and CAF to DNA into two competing processes, viz. hetero-association of the antibiotic-CAF (‘interceptor’ action of CAF) and CAF–DNA complexation (‘protector’ action of CAF). It is found that the complexation of DAU with DNA in the presence of CAF is mainly affected by the CAF–DNA complexation, whereas the binding of either DOX or NOG to DNA is affected approximately equally by both the CAF–DNA complexation and CAF-antibiotic hetero-association. Quantitative evaluation of the three-component mixture of drug–CAF–DNA has enabled the proportion of the antibiotic displaced from DNA on addition of CAF to be calculated over a large range of CAF concentration, which may provide a quantitative basis for the change in anthracycline-related toxicity on addition of CAF.     

X-ray and NMR studies of the DNA oligomer d(ATATAT): Hoogsteen base pairing in duplex DNA

We present and analyze the structure of the oligonucleotide d(ATATAT) found in two different forms by X-ray crystallography and in solution by NMR. We find that in both crystal lattices the oligonucleotide forms an antiparallel double helical duplex in which base pairing is of the Hoogsteen type. The double helix is apparently very similar to the standard B-form of DNA, with about 10 base pairs per turn. However, the adenines in the duplex are flipped over; as a result, the physicochemical features of both grooves of the helix are changed. In particular, the minor groove is narrow and hydrophobic. On the other hand, d(ATATAT) displays a propensity to adopt the B conformation in solution. These results confirm the polymorphism of AT-rich sequences in DNA. Furthermore, we show that extrahelical adenines and thymines can be minor groove binders in Hoogsteen DNA.     

Solution structure of duplex DNA containing an extrahelical abasic site analog determined by NMR spectroscopy and molecular dynamics.

Translesional DNA synthesis past abasic sites proceeds with the preferential incorporation of dAMP opposite the lesion and, depending on the sequence context, one or two base deletions. High-resolution NMR spectroscopy and molecular dynamics simulations were used to determine the three-dimensional structure of a DNA heteroduplex containing a synthetic abasic site (tetrahydrofuran) residue positioned in a sequence that promotes one base deletions. Analysis of NMR spectra indicates that the stem region of the duplex adopts a right-handed helical structure and the glycosidic torsion angle is in anti orientation for all residues. NOE interactions establish Watson-Crick alignments for all canonical base pairs of the duplex. Measurement of distance interactions at the lesion site shows the abasic residue excluded from the helix. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. These structures show a regular duplex region and a slight bend at the lesion site. The tetrahydrofuran residue extrudes from the helix and is highly flexible. The model reported here, in conjunction with a previous study performed on abasic sites, explains the structural bias of one-base deletion mutations.     

Intercalation of daunomycin into d(CG)4 oligomer duplex containing G x T mismatches by vibrational circular dichroism and infrared absorption spectroscopy

The vibrational circular dichroism (VCD) and infrared absorption (IR) spectra of the mismatched octamer oligonucleotides d(CGTGCGCG)(2) (CGT) and d(CGCGTGCG)(2) (CGC) and their complexes with the antitumor drug daunomycin were measured in D(2)O, interpreted, and compared to the octamer d(CGCGCGCG)(2) (CG). The IR spectra of the mismatched octamers in the carbonyl-stretching region are similar to those of the parent CG, whereas the VCD spectra differ in several respects between each other. The main VCD feature due to carbonyl stretching is informative for the mismatches and CG. Vibrational modes in the sugar-phosphate region remain essentially unchanged especially for PO(2) (-) symmetric stretching. Differences between the free and complexed mismatch octamers occurred mainly in the carbonyl-stretching region (1,700-1,600 cm(-1)). The absorption intensity of the C==O peak of G is more prominent for CGC than CGT and resembles CG in this respect. The detailed composition of this doublet is clearly visible, indicating the geometric rearrangement of the base pairs in the presence of the mismatch and upon forming the daunomycin complex.     

The composition of 2-keto aldoses in organic solvents as determined by NMR spectroscopy

The composition of the 2-keto aldoses D-glucosone (1), 6-deoxy-D-glucosone (2), D-allosone (3), and D-galactosone (4) in organic solvents has been determined using NMR spectroscopy. Whereas these keto aldoses form mixtures with up to 15 different isomers in water, the number of forms is significantly decreased in organic solvents. Equilibrium mixtures of 1, 2, and 4 in Me(2)SO, DMF, and pyridine consist to 70-90% of the prevailing alpha-1,5-pyranose form. Two bicyclic forms with a proportion of 80% are the main isomers of 3 in pyridine. Generally, forms with non-hydrated keto functions prevail in non-aqueous solutions.     

Hydration of the RNA duplex r(CGCAAAUUUGCG)2 determined by NMR.

The so-called spine of hydration in the minor groove of AnTn tracts in DNA is thought to stabilise the structure, and kinetically bound water detected in the minor groove of such DNA species by NMR has been attributed to a narrow minor groove [Liepinsh, E., Leupin, W. and Otting, G. (1994) Nucleic Acids Res. 22, 2249-2254]. We report here an NMR study of hydration of an RNA dodecamer which has a wide, shallow minor groove. Complete assignments of exchangeable protons, and a large number of non-exchangeable protons in r(CGCAAAUUUGCG)2 have been obtained. In addition, ribose C2'-OH resonances have been detected, which are probably involved in hydrogen bonds. Hydration at different sites in the dodecamer has been measured using ROESY and NOESY experiments at 11.75 and 14.1 T. Base protons in both the major and minor grooves are in contact with water, with effective correlation times for the interaction of approximately 0.5 ns, indicating weak hydration, in contrast to the hydration of adenine C2H in the homologous DNA sequence. NOEs to H1' in the minor groove are consistent with hydration water present that is not observed in the analogous DNA sequence. Hydration kinetics in nucleic acids may be determined by chemical factors such as hydrogen-bonding more than by simple conformational factors such as groove width.     

Hetero-association of caffeine and aromatic drugs and their competitive binding with a DNA oligomer

NMR spectroscopy has been used to elucidate the molecular basis of the action of caffeine (CAF) on the complexation with DNA of mutagens such as ethidium bromide, propidium iodide, proflavine and acridine orange, and anticancer drugs such as actinomycin D and daunomycin. The hetero-association of CAF and each of the aromatic ligands in 0.1 mol L(-1) phosphate buffer (pD=7.1) has been investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy and analysed in terms of a statistical-thermodynamic model, in which molecules form indefinite aggregates for both self-association and hetero-association. The analysis leads to determination of the equilibrium constants of hetero-association and to the values of the limiting chemical shifts of the heteroassociation of CAF with each of the aromatic molecules. The hetero-association constants between CAF and each of the aromatic drugs/dyes are found to be intermediate in magnitude between those for self-association of CAF and the corresponding drug/dye. The most probable structures of the 1:1 CAF + ligand hetero-association complexes have been determined from the calculated values of the induced limiting chemical shifts of the drug protons. Knowledge of the equilibrium constants for self-association of CAF and the aromatic ligands, for their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the CAF-ligand and CAF-ligand-d(TGCA) complexes to be calculated as a function of CAF concentration in mixed solutions. It is concluded that, on addition of CAF to the solution, the decrease in binding of drug or mutagen with DNA is due both to competition for the binding sites by CAF and the aromatic molecules, and to formation of CAF-ligand hetero-association complexes in the mixed solution; the relative importance of each process depends on the drug or mutagen being considered.     

Compensating bends in a 16-base-pair DNA oligomer containing a T(3)A(3) segment: A NMR study of global DNA curvature

In-phase ligated DNA containing T(n)A(n) segments fail to exhibit the retarded polyacrylamide gel electrophoresis (PAGE) migration observed for in-phase ligated A(n)T(n) segments, a behavior thought to be correlated with macroscopic DNA curvature. The lack of macroscopic curvature in ligated T(n)A(n) segments is thought to be due to cancellation of bending in regions flanking the TpA steps. To address this issue, solution-state NMR, including residual dipolar coupling (RDC) restraints, was used to determine a high-resolution structure of [d(CGAGGTTTAAACCTCG)2], a DNA oligomer containing a T3A3 tract. The overall magnitude and direction of bending, including the regions flanking the central TpA step, was measured using a radius of curvature, Rc, analysis. The Rc for the overall molecule indicated a small magnitude of global bending (Rc = 138 +/- 23 nm) towards the major groove, whereas the Rc for the two halves (72 +/- 33 nm and 69 +/- 14 nm) indicated greater localized bending into the minor groove. The direction of bending in the regions flanking the TpA step is in partial opposition (109 degrees), contributing to cancellation of bending. The cancellation of bending did not correlate with a pattern of roll values at the TpA step, or at the 5' and 3' junctions, of the T3A3 segment, suggesting a simple junction/roll model is insufficient to predict cancellation of DNA bending in all T(n)A(n) junction sequence contexts. Importantly, Rc analysis of structures refined without RDC restraints lacked the precision and accuracy needed to reliably measure bending.     

Novel solution conformation of DNA observed in d(GAATTCGAATTC) by two-dimensional NMR spectroscopy

Resonance assignments of nonexchangeable base and sugar protons of the self-complementary dodecanucleotide d(GAATTCGAATTC) have been obtained by using the two-dimensional Fourier transform NMR methods correlated spectroscopy and nuclear Overhauser effect spectroscopy. Conformational details about the sugar pucker, the glycosidic dihedral angle, and the overall secondary structure of the molecule have been derived from the relative intensities of cross peaks in the two-dimensional NMR spectra in aqueous solution. It is observed that d(GAATTCGAATTC) assumes a novel double-helical structure. The solution conformations of the two complementary strands are identical, unlike those observed in a related sequence in the solid state. Most of the five-membered sugar rings adopt an unusual O1'-endo geometry. All the glycosidic dihedral angles are in the anti domain. The AATT segments A2-T5 and A8-T11 show better stacking compared to the rest of the molecule. These features fit into a right-handed DNA model for the above two segments, with the sugar geometries different from the conventional ones. There are important structural variations in the central TCG portion, which is known to show preferences for DNase I activity, and between G1-A2 and G7-A8, which are cleavage points in the EcoRI recognition sequence. The sugar puckers for G1 and G7 are significantly different from the rest of the molecule. Further, in the three segments mentioned above, the sugar phosphate geometry is such that the distances between protons on adjacent nucleotides are much larger than those expected for a right-handed DNA. We suggest that such crevices in the DNA structure may act as "hot points" in initiation of protein recognition.     

Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy

The broad spectrum of antibacterial activities of host defense cationic antimicrobial peptides (AMPs) arises from their ability to perturb membrane integrity of the microbes. The mechanisms are often thought to require assembly of AMPs on the membrane surface to form pores. However, three dimensional structures in the oligomeric form of AMPs in the context of lipid membranes are largely limited. Here, we demonstrate that a 22-residue antimicrobial peptide, termed VK22, derived from fowlicidin-1, a cathelicidin family of AMP from chicken oligomerizes into a predominantly tetrameric state in zwitterionic dodecylphosphocholine (DPC) micelles. An ensemble of NMR structures of VK22 determined in 200mM perdeuterated DPC, from 755 NOE constrains including 19 inter-helical NOEs, had revealed an assembly of four helices arranged in anti-parallel fashion. Hydrogen bonds, C(α)H-O=C types, and van der Waals interactions among the helical sub-units appear to be involved in the stabilization of the quaternary structures. The central region of the barrel shaped tetrameric bundle is non-polar with clusters of aromatic residues, whereas all the cationic residues are positioned at the termini. Paramagnetic spin labeled NMR experiments indicated that the tetrameric structure is embedded into micelles such that the non-polar region located inside the lipid acyl chains. Structure and micelle localization of a monomeric version, obtained from substitution of two Tyr residues with Ala, of the peptide is also compared. The mutated peptide VK22AA has been found be localized at the surface of the micelles. The tetrameric structure of VK22 delineates a small water pore that can be larger in the higher order oligomers. As these results provide structural insights, at atomic resolution, into the oligomeric states of a helical AMP in lipid environment, the structural details may be further utilized for the design of novel self-assembled membrane protein mimics.     

Solution structure of human Mts1 (S100A4) as determined by NMR spectroscopy

Mts1 is a member of the S100 family of Ca2+-binding proteins and is implicated in promoting tumor progression and metastasis. To better understand the structure-function relationships of this protein and to begin characterizing its Ca2+-dependent interaction with protein binding targets, the three-dimensional structure of mts1 was determined in the apo state by NMR spectroscopy. As with other S100 protein family members, mts1 is a symmetric homodimer held together by noncovalent interactions between two helices from each subunit (helices 1, 4, 1', and 4') to form an X-type four-helix bundle. Each subunit of mts1 has two EF-hand Ca2+-binding domains: a pseudo-EF-hand (or S100-hand) and a typical EF-hand that are brought into proximity by a small two-stranded antiparallel beta-sheet. The S100-hand is formed by helices 1 and 2, and is similar in conformation to other members of the S100 family. In the typical EF-hand, the position of helix 3 is similar to that of another member of the S100 protein family, calcyclin (S100A6), and less like that of other S100 family members for which three-dimensional structures are available in the calcium-free state (e.g., S100B and S100A1). The differences in the position of helix 3 in the apo state of these four S100 proteins are likely due to variations in the amino acid sequence in the C-terminus of helix 4 and in loop 2 (the hinge region) and could potentially be used to subclassify the S100 protein family.     

Geometry and intrinsic tilt of a tryptophan-anchored transmembrane alpha-helix determined by (2)H NMR

We used solid-state deuterium NMR spectroscopy and an approach involving geometric analysis of labeled alanines (GALA method) to examine the structure and orientation of a designed synthetic hydrophobic, membrane-spanning alpha-helical peptide in phosphatidylcholine (PC) bilayers. The 19-amino-acid peptide consists of an alternating leucine and alanine core, flanked by tryptophans that serve as interfacial anchors: acetyl-GWW(LA)(6)LWWA-ethanolamine (WALP19). A single deuterium-labeled alanine was introduced at different positions within the peptide. Peptides were incorporated in oriented bilayers of dilauroyl- (di-C12:0-), dimyristoyl- (di-C14:0-), or dioleoyl- (di-C18:1(c)-) phosphatidylcholine. The NMR data fit well to a WALP19 orientation characterized by a distinctly nonzero tilt, approximately 4 degrees from the membrane normal, and rapid reorientation about the membrane normal in all three lipids. Although the orientation of WALP19 varies slightly in the different lipids, hydrophobic mismatch does not seem to be the dominant factor causing the tilt. We suggest rather that the peptide itself has an inherently preferred tilted orientation, possibly related to peptide surface characteristics or the disposition of tryptophan indole anchors relative to the lipids, the peptide backbone, and the membrane/water interface. Additionally, the data allow us to define more precisely the local alanine geometry in this membrane-spanning alpha-helix.