Solution structure of the chromomycin-DNA complex

The structure of the chromomycin-DNA complex at the deoxyoctanucleotide duplex level has been determined from one- and two-dimensional proton NMR studies in Mg-containing aqueous solution. The NMR results demonstrate that the antitumor agent binds as a symmetrical dimer to the self-complementary d[T-T-G-G-C-C-A-A] duplex with retention of the 2-fold symmetry in the complex. A set of intermolecular nuclear Overhauser enhancements (NOEs) establishes that two chromomycin molecules in the dimer share the minor groove at the G-G-C-C.G-G-C-C segment in such a way that each hydrophilic edge of the chromophore is located next to the G-G.C-C half-site and each C-D-E trisaccharide chain extends toward the 3'-direction of the octanucleotide duplex. In addition, the A-B disaccharide segment and the hydrophilic side chain of the antitumor agent are directed toward the phosphate backbone. The observed changes in nucleic acid NOEs and coupling patterns on complex formation establish a transition to a wider and shallower minor groove at the central G-G-C-C.G-G-C-C segment required for accommodating the chromomycin dimer. The present demonstration that chromomycin binds as a dimer and switches the conformation of the DNA at its G.C-rich minor groove binding site provides new insights into antitumor agent design and the sequence specificity of antitumor agent-DNA recognition.     

Energetics and stereochemistry of DNA complexation with the antitumor AT specific intercalators tilorone and m-AMSA.

Computations by the SIBFA method on the intercalative interaction energies of tilorone and m-AMSA with B-DNA representative oligonucleotides account for the specificity of these antitumor drugs for AT sites and minor groove intercalation. In tilorone this specificity is due to the strong preference of the side chains for the minor groove, which overcomes the preference of the chromophore for a GC intercalation site. In m-AMSA the specificity is due to the combined preference of both the chromophore and the anilino side chain for AT intercalation site and minor groove, respectively. o-AMSA is shown to manifest a similar (although significantly less pronounced specificity) as m-AMSA but a higher affinity for DNA. A comparison of the energetics and stereochemistry of intercalative binding to DNA of m-AMSA (AT minor groove specific) and 9-aminoacridine-4-carboxamide (GC major groove specific), which possess the same chromophore and differ only by the nature and position of the side chains, shows the possibility of important variations in the intercalative behaviour of chromophoric drugs as a function of the substituent groups attached to them.     

A high-resolution structure of a DNA-chromomycin-Co(II) complex determined from pseudocontact shifts in nuclear magnetic resonance

BACKGROUND: The drug chromomycin-A(3) binds to the minor groove of DNA and requires a divalent metal ion for complex formation. (1)H, (31)P and (13)C pseudocontact shifts occurring in the presence of a tightly bound divalent cobalt ion in the complex between d(TTGGCCAA)(2) and chromomycin-A(3) have been used to determine the structure of the complex. The accuracy of the structure was verified by validation with nuclear Overhauser enhancements (NOEs) and J-coupling constants not used in the structure calculation. RESULTS: The final structure was determined to 0.7 A resolution. The structure was compared with a structure obtained in an earlier study using NOEs, in order to assess the accuracy of NOEs in giving global structural information for a DNA complex. Although some basic features of the structures agreed, they differed substantially in the fine structural details and in the DNA axis curvature generated by the drug. The distortion of base-pair planarity that was observed in the NOE structure was not seen in our structure. Differences in drug orientation and hydrogen bonding also occurred. The curvature and elongation of the DNA that was obtained previously was not found to occur in our study. CONCLUSIONS: The use of pseudocontact shifts has enabled us to obtain a high-precision global structure of the chromomycin-DNA complex, which provides an accurate template on which to consider targeting minor groove binding drugs. The effect of such binding is not propagated far along the helix but is restricted to a local kink in the axis that reverts to its original direction within four base pairs.     

Comparative thermodynamics for monomer and dimer sequence-dependent binding of a heterocyclic dication in the DNA minor groove

Phenylamidine cationic groups linked by a furan ring (furamidine) and related symmetric diamidine compounds bind as monomers in the minor groove of AT sequences of DNA. DB293, an unsymmetric derivative with one of the phenyl rings of furamidine replaced with a benzimidazole, can bind to AT sequences as a monomer but binds more strongly to GC-containing minor-groove DNA sites as a stacked dimer. The dimer-binding mode has high affinity, is highly cooperative and sequence selective. In order to develop a better understanding of the correlation between structural and thermodynamic aspects of DNA molecular recognition, DB293 was used as a model to compare the binding of minor-groove agents with AT and mixed sequence DNA sites. Isothermal titration calorimetry and surface plasmon resonance results clearly show that the binding of DB293 and other related compounds into the minor groove of AT sequences is largely entropy-driven while the binding of DB293 as a dimer into the minor groove of GC-containing sequences is largely enthalpy-driven. At 25 degrees C, for example, the AT binding has DeltaG degrees, DeltaH degrees and TDeltaS degrees values of -9.6, -3.6 and 6.0 kcal/mol while the values for dimer binding to a GC-containing site are -9.0, -10.9 and -1.9 kcal/mol (per mol of bound compound), respectively. These results show that the thermodynamic components for binding of compounds of this type to DNA are very dependent on the structure, solvation and sequence of the DNA binding site.     

Assembling the human IFN-beta enhanceosome in solution

Assembly of interferon-β enhanceosome from its individual protein components and of enhancer DNA has been studied in solution using a combination of fluorescence anisotropy, microcalorimetry, and CD titration. It was shown that the enhancer binds only one full-length phosphomimetic IRF-3 dimer at the PRDIII-PRDI sites, and this binding does not exhibit cooperativity with binding of the ATF-2/c-Jun bZIP (leucine zipper dimer with basic DNA recognition segments) heterodimer at the PRDIV site. The orientation of the bZIP pair is, therefore, not determined by the presence of the IRF-3 dimer, but is predetermined by the asymmetry of the PRDIV site. In contrast, bound IRF-3 dimer interacts strongly with the NF-κB (p50/p65) heterodimer bound at the neighboring PRDII site. The orientation of bound NF-κB is also predetermined by the asymmetry of the PRDII site and is the opposite of that found in the crystal structure. The HMG-I/Y protein, proposed as orchestrating enhanceosome assembly, interacts specifically with the PRDII site of the interferon-β enhancer by inserting its DNA-binding segments (AT hooks) into the minor groove, resulting in a significant increase in NF-κB binding affinity for the major groove of this site.     

Minor groove to major groove, an unusual DNA sequence-dependent change in bend directionality by a distamycin dimer

DNA sequence-dependent conformational changes induced by the minor groove binder, distamycin, have been evaluated by polyacrylamide gel electrophoresis. The distamycin binding affinity, cooperativity, and stoichiometry with three target DNA sequences that have different sizes of alternating AT sites, ATAT, ATATA, and ATATAT, have been determined by mass spectrometry and surface plasmon resonance to help explain the conformational changes. The results show that distamycin binds strongly to and bends five or six AT base pair minor groove sites as a dimer with positive cooperativity, while it binds to ATAT as a weak, slightly anticooperative dimer. The bending direction was evaluated with an in phase A-tract reference sequence. Unlike other similar monomer minor groove binding compounds, such as netropsin, the distamycin dimer changes the directionality of the overall curvature away from the minor groove to the major groove. This distinct structural effect may allow designed distamycin derivatives to have selective therapeutic effects.     

Structure refinement of the chromomycin dimer-DNA oligomer complex in solution.

We have refined the initial docking model of the Mg(II)-co-ordinated chromomycin-d(A2G2C2T2) complex (2 drug equivalents per duplex) by a complete relaxation matrix analysis simulation of the two-dimensional nuclear Overhauser effect (NOESY) spectrum of the complex in 2H2O solution. This relaxation matrix refined structure of the complex exhibits the following characteristics. (1) We observe an unwound and elongated duplex that exhibits characteristics distinct from the A and B-DNA family of helices at the central (G-G-C-C).(G-G-C-C) chromomycin dimer binding and flanking sites. On the other hand sugar puckers, glycosidic torsion angles, displacement of the base-pairs from the helix axis and the minor groove width for this central tetranucleotide segment all fall within the A-family of helical parameters. (2) The chromomycin monomers are aligned in a head-to-tail orientation in the Mg(II)-co-ordinated dimer in the complex. The chromophores are aligned with a slight tilt relative to each other and make an angle of 75 degrees between their planes. The C-D-E trisaccharide segments from individual monomers adopt an extended conformation that projects in opposite directions in the dimer. The divalent metal cation is co-ordinated to the O(1) carbonyl and O(9) enolate atoms of the chromophores and aligns them such that the O(9)-Mg-O(9) angle is 170 degrees while all other O-Mg-O angles are in the 95(+/- 15)degrees range. (3) The sequence specificity of the chromomycin dimer for the widened and shallower (G3-G4-C5-C6).(G3-G4-C5-C6) minor groove binding site is associated with intermolecular hydrogen bonds formed between the OH group at C(8) of the chromophore and the minor groove NH2 group at position 2 and N(3) groups of G4 and between the O(1) oxygen of the E-sugar and the minor groove NH2 group at position 2 of G3 in the complex. (4) Additional intermolecular interactions are primarily van der Waals contacts between anomeric and adjacent CH2 protons on each sugar in the C-D-E trisaccharide segments of the chromomycin dimer and the minor groove surface of the DNA. These results provide insights into the induced conformational transitions required to generate a complementary match between the drug dimer and its DNA binding site on complex formation.     

DNA minor groove binding agents interfere with topoisomerase II mediated lesions induced by epipodophyllotoxin derivative VM-26 and acridine derivative m-AMSA in nuclei from L1210 cells

This study demonstrated that agents capable of interacting with the minor groove in nuclear DNA interfere with topoisomerase II mediated effects of antitumor drugs such as VM-26 and m-AMSA. Distamycin, Hoechst 33258, and DAPI were used as agents capable of AT-specific binding in the minor groove of DNA while producing no profound long-range distortion of DNA structure. In intact nuclei from L1210 cells, these minor groove binders inhibited the induction of topoisomerase II mediated DNA damage (DNA-protein cross-links and DNA double-strand breaks) by VM-26 and m-AMSA. The inhibitory effects of distamycin reflected prevention of formation of new lesions but not reversal of preexisting damage. The minor groove binders did not differentiate between lesions induced by an intercalator, m-AMSA, or by a DNA-nonbinding drug, VM-26. All three groove binders inhibited DNA breaks more strongly than DNA-protein cross-links. The inhibitory potency correlated with the size of minor groove binders and the size of their DNA-binding sites: distamycin (5 bp) greater than Hoechst 33258 (4 bp) greater than DAPI (3 bp). The results showed that DNA minor groove binders are a new type of modulators of the action of topoisomerase II targeted drugs.     

Ternary interactions of spermine with DNA: 4''-epiadriamycin and other DNA: anthracycline complexes.

The recently developed anthracycline 4'-epiadriamycin, an anti-cancer drug with improved activity, differs from adriamycin by inversion of the stereochemistry at the 4'-position. We have cocrystallized 4'-epiadriamycin with the DNA hexamer d(CGATCG) and solved the structure to 1.5 A resolution using x-ray crystallography. One drug molecule binds at each d(CG) step of the hexamer duplex. The anthracycline sugar binds in the minor groove. A feature of this complex which distinguishes it from the earlier DNA:adriamycin complex is a direct hydrogen bond from the 4'-hydroxyl group of the anthracycline sugar to the adenine N3 on the floor of the DNA minor groove. This hydrogen bond results directly from inversion of the stereochemistry at the 4'-position. Spermine molecules bind in the major groove of this complex. In anthracycline complexes with d(CGATCG) a spermine molecule binds to a continuous hydrophobic zone formed by the 5-methyl and C6 of a thymidine, C5 and C6 of a cytidine and the chromophore of the anthracycline. This report discusses three anthracycline complexes with d(CGATCG) in which the spermine molecules have different conformations yet form extensive van der Waals contacts with the same hydrophobic zone. Our results suggest that these hydrophobic interactions of spermine are DNA sequence specific and provide insight into the question of whether DNA:spermine complexes are delocalized and dynamic or site-specific and static.     

Minor groove deformability of DNA: a molecular dynamics free energy simulation study

The conformational deformability of nucleic acids can influence their function and recognition by proteins. A class of DNA binding proteins including the TATA box binding protein binds to the DNA minor groove, resulting in an opening of the minor groove and DNA bending toward the major groove. Explicit solvent molecular dynamics simulations in combination with the umbrella sampling approach have been performed to investigate the molecular mechanism of DNA minor groove deformations and the indirect energetic contribution to protein binding. As a reaction coordinate, the distance between backbone segments on opposite strands was used. The resulting deformed structures showed close agreement with experimental DNA structures in complex with minor groove-binding proteins. The calculated free energy of minor groove deformation was approximately 4-6 kcal mol(-1) in the case of a central TATATA sequence. A smaller equilibrium minor groove width and more restricted minor groove mobility was found for the central AAATTT and also a significantly ( approximately 2 times) larger free energy change for opening the minor groove. The helical parameter analysis of trajectories indicates that an easier partial unstacking of a central TA versus AT basepair step is a likely reason for the larger groove flexibility of the central TATATA case.     

Effects of DNA structure on oxopropenylation by the endogenous mutagens malondialdehyde and base propenal

Malondialdehyde (MDA) and nucleobase propenals can transfer oxopropenyl groups to guanine residues of DNA to yield pyrimodopurinone (M(1)G) adducts. The DNA structural requirements for reaction with alpha,beta-unsaturated aldehydes were explored. We found that single-stranded DNA is more sensitive to oxopropenylation than double-stranded DNA, and supercoiled plasmid DNA is more sensitive than linearized plasmid DNA. Increasing ionic strength inhibits oxopropenylation, especially by adenine propenal. The intercalating agents ethidium bromide and 9-aminoacridine enhanced oxopropenylation by severalfold. In contrast, actinomycin D, which both intercalates and binds in the minor groove, inhibited oxopropenylation. The anthracycline drugs daunorubicin and doxorubicin enhanced oxopropenylation by MDA up to 3-fold and by adenine propenal up to 7-fold in a concentration-dependent manner. The minor groove binders netropsin and distamycin inhibited oxopropenylation, but methyl green, a major groove binder, had little effect. These data suggest that steric access to the target nucleophile located in the minor groove of DNA is critical for adduct formation by the endogenous mutagens MDA and base propenals.     

The A.T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure.

We have determined the domains of the mammalian high mobility group (HMG)I chromosomal proteins necessary and sufficient for binding to the narrow minor groove of stretches of A.T-rich DNA. Three highly conserved regions within each of the known HMG-I proteins is closely related to the consensus sequence T-P-K-R-P-R-G-R-P-K-K. A synthetic oligopeptide corresponding to this consensus "binding domain" (BD) sequence specifically binds to substrate DNA in a manner similar to the intact HMG-I proteins. Molecular Corey-Pauling-Koltun model building and computer simulations employing energy minimization programs to predict structure suggest that the consensus BD peptide has a secondary structure similar to the antitumor and antiviral drugs netropsin and distamycin and to the dye Hoechst 33258. In vitro these ligands, which also preferentially bind to A.T-rich DNA, have been demonstrated to effectively compete with both the BD peptide and the HMG-I proteins for DNA binding. The BD peptide also contains novel structural features such as a predicted Asx bend or "hook" at its amino-terminal end and laterally projecting cationic Arg/Lys side chains or "bristles" which may contribute to the binding properties of the HMG-I proteins. The predicted BD peptide structure, which we refer to as the "A.T-hook," represents a previously undescribed DNA-binding motif capable of binding to the minor groove of stretches of A.T base pairs.     

Quantitative footprinting analysis of the chromomycin A3--DNA interaction.

Chromomycin A3 (CHR) binding to the duplex d(CAAGTCTGGCCATCAGTC).d(GACTGATGGCCAGACTTG) has been studied using quantitative footprinting methods. Previous NMR studies indicated CHR binds as a dimer in the minor groove. Analysis of autoradiographic spot intensities derived from DNase I cleavage of the 18-mer in the presence of various amounts of CHR revealed that the drug binds as a dimer to the sequence 5'-TGGCCA-3',3'-ACCGGT-5' in the 18-mer with a binding constant of (2.7 +/- 1.4) x 10(7) M-1. Footprinting and fluorescence data indicate that the dimerization constant for the drug in solution is approximately 10(5) M-1. Since it has been suggested that CHR binding alters DNA to the A configuration, quantitative footprinting studies using dimethyl sulfate, which alkylates at N-7 of guanine in the major groove, were also carried out. Apparently, any drug-induced alteration in DNA structure does not affect cleavage by DMS enough to be observed by these experiments.