Molecular Structure,Conformation and Interactions of Antitumor Antibiotic Cyanonaphthridinomycin,a Covalent Binder of DNA

Abstract

X-ray, NMR and molecular modeling studies on cyanonaphthridinomycin (C₂₂ H₂₆N₄O₅), a DNA binding antibiotic, have been carried out to study the structure, conformation and interactions with DNA. The crystals belong to the space group P₂₁ with the cell dimensions of a = 5.934(1), b = 20.684(4), c = 16.866(3)A γ = 90.9° and Z = 4(two molecules/asymmetric unit). The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.087 for 4061 reflections. The conformation of the molecule is compared with that of naphthridinomycin. There are differences in the orientation of the methoxyl group and the saturated oxazole ring. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. Molecular mechanics studies were carried out to obtain the energy minimized structure and its comparison with X-ray and NMR results. Molecular modelling studies were performed to propose models for drug-DNA interactions. Both partial intercalation and groove-binding models have been proposed.     

Stereochemistry of Actinomycin–DNA Binding

The three dimensional structure of a crystalline complex containing actino-mycin D and deoxyguanosine has shed light on the stereochemistry of actino-mycin binding to DNA. The phenoxa-zone ring system of actinomycin intercalates into the DNA helix, while deoxyguanosine residues interact with both cyclic peptides through specific hydrogen bonds.     

抑制启动子的三链DNA的结构模建及稳定性研究

在IRIS Indigo2(SGI公司)工作站上,利用InsightⅡ/MSI软件包,以TAT三链DNA为模板,采用同源模建的方法,分别建立起两个含21nt的脱氧寡核苷酸CP1(G3TG2TGT2G5TG2TGT)和CP3(TGTG2TG5T2GTG2TG3)的三维结构.采用分子力学方法进行能量优化,将得到的能量最低结构作为分子的优势构象.研究结果显示,CP1的能量低于CP3的能量,即前者的结构较后者稳定.从而证明了CP1与乙肝病毒（HBV）的核心启动子（Cp）片段之间能稳定地形成三链DNA,并能特异性地抑制DNA结合蛋白与Cp片段的结合.这些结果表明,三链DNA的形成有可能抑制DNA的转录.     

Molecular Mechanisms of Tight Binding through Fuzzy Interactions

Many intrinsically disordered proteins (IDPs) form fuzzy complexes upon binding to their targets. Although many IDPs are weakly bound in fuzzy complexes, some IDPs form high-affinity complexes. One example is the nonstructural protein 1 (NS1) of the 1918 Spanish influenza A virus, which hijacks cellular CRKII through the strong binding affinity (K_d ～10 nM) of its proline-rich motif (PRM^NS1) to the N-terminal Src-homology 3 domain of CRKII. However, its molecular mechanism remains elusive. Here, we examine the interplay between structural disorder of a bound PRM^NS1 and its long-range electrostatic interactions. Using x-ray crystallography and NMR spectroscopy, we found that PRM^NS1 retains substantial conformational flexibility in the bound state. Moreover, molecular dynamics simulations showed that structural disorder of the bound PRM^NS1 increases the number of electrostatic interactions and decreases the mean distances between the positively charged residues in PRM^NS1 and the acidic residues in the N-terminal Src-homology 3 domain. These results are analyzed using a polyelectrostatic model. Our results provide an insight into the molecular recognition mechanism for a high-affinity fuzzy complex.     

乳酸链球菌素的分子结构、抗菌活性及基因工程研究

乳酸链球菌素(Nisin)是一种由34个氨基酸组成的天然抗菌素,对革兰氏阳性菌具有广谱抑菌作用。Nisin通过吸附于细胞膜,在膜上形成孔洞杀菌。Nisin的11个基因形成基因簇,负责其翻译后修饰、转运,先导序列的切除,成熟分子的外泌,合成过程的调节等。Nisin高产菌株的选育主要通过物理化学诱变,基因工程高效表达技术的研究刚刚开始。Nisin在食品和医药领域具有广泛的应用。本文主要就Nisin的分子结构、分泌机制、抗菌活性、作用机理,特别是它的基因工程技术研究进展等作一概述。     

Molecular Interactions of Etazolate with Benzodiazepine and Picrotoxinin Binding Sites

Pyrazolopyridines, such as etazolate (SQ 20009), enhance [3H]diazepam binding to a Lubrol-solubilized fraction that has specific binding sites for 3H benzodiazepines, [alpha-3H]dihydropicrotoxinin (DHP) and [3H]muscimol. Etazolate enhancement of [3H]diazepam binding was inhibited by picrotoxinin. Furthermore, etazolate inhibited the [3H]DHP binding in a Lubrol-solubilized fraction with an IC50 value of 6-8 microM. These results provide evidence that etazolate, like pentobarbital, modulates benzodiazepine binding via the DHP-sensitive site of the benzodiazepine-GABA receptor-ionophore complex.     

FTIR Study of Specific Binding Interactions Between DNA Minor Groove Binding Ligands and Polynucleotides

Abstract

The use of FTIR spectroscopy is made to study the interactions between polynucleotides and two series of minor groove binding compounds. The latter were developed and described previously as part of an ongoing program of rational design of modified Ligands based on naturally occurring pyrrole amidine antibiotic netropsin, and varying the structure of bis- benzimidazole chromosomal stain Hoechst 33258. Characteristic IR absorptions due to the vibrations of thymidine and cytosine keto groups in polynucleotides containing AT and GC base pairs respectively are used to monitor their interaction with the added Ligands. Although the two thiazole based lexitropsins based on netropsin structure differ in the relative orientation of nitrogen and sulfur atoms with respect to the concave edge of the molecules, they interact exclusively with the thymidine C2=O carbonyl groups in the minor groove of the alternating AT polymer as evidenced by specific changes in the IR spectra.

In the second series of compounds based on Hoechst 33258, the structure obtained by replacing the two benzimidazoles in the parent compound by a combination of pyridoimidazole and benzoxazole, exhibits changes in the carbonyl frequency region of poly dG · poly dC which is attributed to the ligand interaction at the minor groove of GC base pairs. In contrast, Hoechst 33258 itself interacts only with poly dA · poly dT. Weak or no interaction exists between the Ligands and any of the polynucleotides at the levels of the phosphate groups or the deoxyribose units.     

Molecular Forces for the Binding and Condensation of DNA Molecules

Atomic force microscopy has been used to investigate the binding between a double-stranded DNA and bilayers of cationic lipids and zwitterionic lipids in low ionic-strength solutions. The binding of a DNA molecule to freshly cleaved mica surface in solution has also been measured. The binding of DNA molecules to cationic lipid bilayers has a minimal strength of ∼45 pN. On zwitterionic lipid bilayers and mica surface, the minimal binding strength is approximately twice that value. The binding also has a dynamic nature, with only a certain percentage of recorded force curves containing the binding characteristics. Divalent Mg²⁺ ions enhance the binding by increasing that percentage without any effect on the binding strength. We have also observed a long-range attraction between DNA molecules and cationic lipid bilayers with a strength much larger than the minimum force and a range well over 50 nm, possibly related to the driving force responsible for the two-dimensional condensation of DNA.     

Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.     

Carcinogen-Nucleic Acid Interactions: Equilibrium Binding Studies of Aflatoxins B1 and B2 with DNA and the Oligodeoxynucleotide d(ATGCAT)2

Abstract

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B₁ and B₂ with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B₂ with the oligodeoxynucleotide d(ATGCAT)₂. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B₁ binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin ? 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels ? 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a “two-site” binding model [L.S. Rosenberg, M J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002–1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B₂ binding to d(ATGCAT)₂ indicates that the aflatoxin B₂/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1–0.5 ppm are observed for the aflatoxin B₂ 4-OCH₃, H5, and H6a protons. Much smaller chemical shift changes ? 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T₁ experiments demonstrate a 15–25 % decrease in the relaxation time for the adenine H2 protons when aflatoxin B₂ is added to the solution. This result suggests that aflatoxin B₂ protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B₁ and B₂ complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B₁,-guanine N7 DNA adducts.     

线虫线粒体DNA结构及分子生物学特性

线粒体DNA含量丰富、分子量小、分子进化速度快、结构基因保守性强,被广泛应用于分子遗传进化、分类鉴定及发育生物学等方面的研究。     

Anthracycline antibiotics. Interaction with DNA and nucleosomes and inhibition of DNA synthesis

We report studies of the interaction of four anthracycline antibiotics, iremycin (IM), daunomycin (DM), aclacinomycin A (AM), and violamycin B1 (VM), with naked DNA, nucleosomal core particles, and 175 base pair (bp) nucleosomes lacking histone H1. In all cases the binding strength increases in the order IM less than DM approximately AM less than VM. The binding substrates increased in affinity for the drugs in the following order: core particles less than 175-bp nucleosomes less than DNA. The apparent DNA length increment per drug bound decreases in the progression IM greater than DM greater than AM greater than VM, the same serial order as is characterized by increasing binding affinity. Dichroism amplitude measurements show that for all drugs the long-wavelength absorbance transition moment is tilted by 26-29 degrees relative to the plane perpendicular to the helix axis; this angle probably corresponds to the long axis tilt of the intercalated chromophore. Finally, it was found that the ability of the drugs to inhibit DNA synthesis by Escherichia coli DNA polymerase I increases in the same order as their binding affinity.     

Mapping the Binding Site of Aflatoxin B1 in DNA: Molecular Modeling of the Binding Sites for the N(7)-Guanine Adduct of Aflatoxin B1 in Different DNA Sequences

Abstract

Aflatoxin B, (AFB₁), a potent mutagen and carcinogen, forms an adduct exclusively at the N(7) position of guanine, but the structure of this adduct in double stranded DNA is not known. Molecular modeling (using the program, PSFRODO) in conjunction with molecular mechanical calculation (using the program, AMBER) are used to assess the binding modes available to this AFB₁ adduct TVvo modes appear reasonable; in one the AFB₁ moiety is intercalated between the base pair containing the adducted guanine and the adjacent base pair on the 5₁-side in reference to the adducted guanine, while in the second it is bound externally in the major groove of DNA Rotational flexibilty appears feasible in the latter providing four, potential binding sites. Molecular modeling reveals that the binding sites around the reactive guanine in different sequences are not uniformly compatible for interaction with AFB₁. As the sequence is changed, one particular external binding site would be expected to give a pattern of reactivities that is reasonably consistent with the observed sequence specificity of binding that AFB₁ shows in its reaction with DNA (Benasutti, M., Ejadi, S., Whitlow, M. D. and Loechler, E. L. (1988) Biochemistry 27, 472–481). The AFB₁ moiety is face-stacked in the major groove with its long axis approximately perpendicular to the helix axis. Favorable interactions are formed between exocyclic amino groups that project into the major groove on cytosines and adenines surrounding the reactive guanine, and oxygens in AFB₁; unfavorable interactions involve van der Waals contacts between the methyl group on thymine and the AFB₁ moiety. “Some of the sequence specificity of binding data can be rationalized more readily if it is assumed that 5′-GG-3′ sequences adopt an A-DNA structure.” Based upon molecular modeling/potential energy minimization calculation, it is difficult to predict how reactivity would change in different DNA sequences in the case of the intercalative binding mode; however, several arguments suggest that intercalation might not be favored. From these considerations a model of the structure for the transition state in reaction of AFB, with DNA is proposed involving one particular external binding site.     

Integrin Structure,Activation, and Interactions

Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large “head” on two “legs,” with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains. These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allow cells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure. This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor.The name “integrin” was suggested for an integral membrane protein complex first characterized in 1986 (Tamkun et al. 1986). The name was devised because the protein identified linked the extracellular matrix to the cytoskeleton (early developments in this field have been well described [Hynes 2004]). In the 25 years since that first characterization, a vast amount of work has been performed, with consequent increased understanding. The essential role of integrins in tissue organization and cell development, their signal transduction mechanisms (from outside to in and inside to out!), and their potential as therapeutic targets is now established. In this article, we provide an overview of the structure of integrins, the conformational changes that determine activation state, and the mechanisms of ligand binding.