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Structural Investigation of the Hedamycin:d(ACCGGT)2 Complex by NMR and Restrained Molecular Dynamics
Biochemical and Biophysical Research Communications 290, 1602–1608 (2002) doi:10.1006/bbrc.2002.6369, available online at http://www.idealibrary.com on
Structural Investigation of the Hedamycin:d(ACCGGT) 2 Complex by NMR and Restrained Molecular Dynamics Elisabeth A. Owen,* Glenn A. Burley,† John A. Carver,† Geoffrey Wickham,† ,1 and Max A. Keniry* ,2 *Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia; and †Department of Chemistry, University of Wollongong, Wollongong, New South Wales 2522, Australia
Received November 26, 2001
Hedamycin, a member of the pluramycin family of drugs, displays a range of biological responses including antitumor and antimicrobial activity. The mechanism of action is via direct interaction with DNA through intercalation between the bases of the oligonucleotide and alkylation of a guanine residue at 5ⴕPyG-3ⴕ sites. There appears to be some minor structural differences between two earlier studies on the interaction of hedamycin with 5ⴕ-PyG-3ⴕ sites. In this study, a high-resolution NMR analysis of the hedamycin:d(ACCGGT) 2 complex was undertaken in order to investigate the effect of replacing the thymine with a guanine at the preferred 5ⴕ-CGT-3ⴕ site. The resultant structure was compared with earlier work, with particular emphasis placed on the drug conformation. The structure of the hedamycin:d(ACCGGT) 2 complex has many features in common with the two previous NMR structures of hedamycin:DNA complexes but differed in the conformation and orientation of the N,Ndimethylvancosamine saccharide of hedamycin in one of these structures. The preferential binding of hedamycin to 5ⴕ-CG-3ⴕ over 5ⴕ-TG-3ⴕ binding sites is explained in terms of the orientation and location of the N,N-dimethylvancosamine saccharide in the minor groove. © 2002 Elsevier Science (USA) Key Words: 2D NMR; drug–DNA; hedamycin; oligonucleotide.
Coordinates: The coordinates of the hedamycin:d(ACCGGT) 2 complex have been deposited with the Protein Data Bank (Accession No. 1JHI). Abbreviations used: NMR, nuclear magnetic resonance; COSY, correlation spectroscopy; NOESY, two-dimensional nuclear Overhauser exchange spectroscopy; PCOSY, purged correlation spectroscopy; TOCSY, total correlation spectroscopy. 1 Present address: Polymerat Pty. Ltd., St Lucia, Queensland 4067, Australia. 2 To whom correspondence and reprint requests should be addressed. Fax: ⫹61 2 6125 0750. E-mail: [email protected]. 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
Hedamycin (Fig. 1) is a naturally occurring antibiotic from the pluramycin family of antibiotics derived from the Streptomyces genera of fungi. Interest in the pluramycins arose from a range of biological responses, such as antitumor and antimicrobial activity (1, 2). Structurally, hedamycin consists of a highly substituted 4H-anthra[1,2-b]-pyran-4,7,12-trione chromophore with two amino sugars, an anglosamine attached to the C8 position and an N,N-dimethyvancosamine attached to the C10 position (Fig. 1) (1). Hedamycin alkylates nucleic acids at guanine sites with high 5⬘-PyGT-3⬘ selectivity but favors 5⬘-CGT-3⬘ sequences (3–5). Substantial adduct formation is also observed at 5⬘-CGG-3⬘ sequences (4). Previous NMR studies have shown that the anthrapyrantrione chromophore of hedamycin intercalates between the 5⬘PyG-3⬘ bases with the two amino sugars placed in the minor groove and the bisepoxide chain located in the major groove (5–7). The anglosamine sugar is located to the 3⬘ side of the intercalation site and the N,Ndimethylvancosamine sugar is located to the 5⬘ side of the intercalation site. The preferential selectivity for a pyrimidine on the 5⬘ side of the alkylated guanine appears to arise from an interaction between the O2 carbonyl of the pyrimidine and the positively charged dimethylamino substituent of the N,N-dimethylvancosamine sugar (6). The preference for cytosine over thymine in this position is less obvious although it has been suggested that more extensive van der Waals interactions between the sugar and the wall of the minor groove favor the cytosine base (6). Here we present an NMR-derived, solution phase, three dimensional model of the hedamycin:d(ACCGGT) 2 complex that was determined by relaxation matrix refinement of the NOESY intensities, followed by torsion angle molecular dynamics and energy minimization. The model of hedamycin:d(ACCGGT) 2 is in substantial agreement with previous NMR studies of hedamycin–DNA complexes (6, 7) but differs in detail with one of these studies (6). The model calls into
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 600 NMR spectrometers using COSY, TOCSY and NOESY spectra. A heteronuclear proton-detected 31P– 1H correlation experiment was collected at 600 MHz on a Varian INOVA 600 spectrometer by the g-HELCO technique (10). Quantitative two-dimensional NOESY spectra were acquired on a Varian INOVA 600 spectrometer. These data included NOESY spectra (80, 140, and 200 ms mixing times, 3.5 s recycle time) of the ⬃1 mM hedamycin-d(ACCGGT) 2 complex in 10 mM phosphate, 50 mM NaCl, in pH 6.7 D 2O buffer at 15°C and a NOESY spectrum (250 ms mixing time) of the complex in a similar 90% H 2O/10% D 2O buffer at 15°C. All spectra were processed with Varian VNMR software (Varian Associates, Palo Alto) and quantitated and visualized using XEASY software (11). Typically 4096 complex points were collected over 5200 Hz in the t 2 domain and 700 complex points over 5200 Hz in the t 1 domain. Datasets were zerofilled to a final size of 4096 ⫻ 2048 complex points prior to Fourier transformation.
FIG. 1. Structure of hedamycin and the DNA sequence showing the intercalation site. The modified guanine is shown in bold.
question the mechanism by which cytosine is favored over thymine at the 5⬘ side of the 5⬘-PyG-3⬘ intercalation site (6). We propose a different mechanism for this selectivity that is consistent with the evolution of a N,N-dimethylvancosamine sugar at the C10 position on the chromophore. Hedamycin clearly interacts with the 5⬘-CGG-3⬘ site by covalent adduct formation at the central guanine position but there appears to be no obvious reason for a preference for thymine at the 3⬘ position in this sequence as indicated by sequence selectivity studies (8). MATERIALS AND METHODS Sample preparation. The hedamycin used in this work was a generous gift from Bristol-Myers Squibb and was used without further purification. d(ACCGGT) was synthesized on an Applied Biosystems Model 391A DNA synthesizer using solid phase phosphoramidite chemistry and purified by reverse phase HPLC followed by extensive dialysis. The drug–DNA adduct was formed by adding 10 l aliquots of a 10 mM solution of hedamycin to a 1.0 mM solution of d(ACCGGT) 2 in 10 mM phosphate, 50 mM NaCl, pH 6.7 D 2O buffer. The titrations were conducted in the dark and nitrogen was bubbled through the reaction mixture after the addition of each drug aliquot to prevent photo-oxidation of the ligand (9). The mixture was characterized after each addition by the acquisition of 1H NMR spectra. After the final addition of hedamycin at a 1:1 ratio of hedamycin: d(ACCGGT) 2 the complex was allowed to stand for 12 h at 25°C to ensure complete reaction. NMR spectroscopy. Initial characterization and assignment of the complex was performed on Varian Unity-400 and Bruker DMX-
Experimental restraints. Initially, crosspeaks between the nonexchangeable protons were divided into strong, medium and weak categories and given bounds of 0.2– 0.35, 0.35– 0.45 and 0.4 – 0.55 nm, respectively. A similar strategy was adopted for the crosspeaks that involved exchangeable protons in the NOESY spectra of the complex in 90% H 2O/10% D 2O buffer. In addition, 16 restraints were created to maintain the hydrogen bonds between the bases of the two nucleotide chains and given the bonds: GC pairs, 0.285– 0.305 nm (G-O6 to C-N4), (G-N1 to C-N3) and 0.276 – 0.296 nm (G-N2 to C-O2); AT pairs, 0.275– 0.295 nm (A-N1 to T-N3) and 0.285– 0.205 nm (A-N6 to T-O4). Backbone dihedral angles were conservatively restrained about 0° ⫾ 150° (␣), 180° ⫾ 60° (), 60° ⫾ 60° (␥) and 0° ⫾ 150° () following analysis of 31P chemical shifts (12), H2⬘-H5⬘/H5⬙ and H6/ H8-H5⬘/H5⬙ NOEs and H3⬘, H4⬘ and H5⬘/H5⬙ linewidths in the NOESY spectra following the procedure of Reid et al. (13, 14). Structure determination and refinement. Interproton distance restraints were estimated from the crosspeak intensity of the 2D NOESY spectra by employing the MARDIGRAS (Version 5.2) program and the pseudoenergy force field was derived from these restraints as described in Keniry et al. (15). The initial model of self-complementary d(ACCGGT) 2 was created from standard A- and B-conformation DNA coordinates using the QUANTA 4.0 Nucleic Acid Builder (MSI, Waltham, MA). The hedamycin structure was generated from the crystal structure coordinates (16). The complex was formed by breaking the epoxide ring containing the C18 carbon and attaching C18 covalently to the N7 of G4 in the sequence d(A1C2-C3-G4-G5-T6). The chromophore was placed between the C3-G10 and G4-C9 base pairs and the two saccharides located on the minor groove side of the oligonucleotide. The initial models were extensively minimized and then subjected to Cartesian space molecular dynamics using the QUANTA/ CHARMm 23.1 package (MSI, Waltham, MA) on a Silicon Graphics O2 workstation. The models were further refined using torsion angle dynamics protocol of CNS (17) against distances derived from crosspeak intensities obtained from NOESY spectra with mixing times of 80, 150, and 200 ms and refined with several iterations of MARDIGRAS. Crosspeak intensities for the exchangeable protons were not subject to relaxation matrix analysis but instead were assigned distance bounds as described above. In all, there were 394 distance restraints. Of these, 307 were estimated from the NOESY volumes and 64 other restraints derived from the NMR spectra that were converted into distance restraints. A version of the standard anneal protocol of CNS, modified to allow up to 60 ps of torsion angle dynamics at 20,000°K was used. The models were cooled from 20,000 to 1,000°K over 76 cycles of 50 steps (each cycle taking 0.75 ps). Final cooling from 1,000 to 300°K was over 70 cycles of 30 steps using Cartesian dynamics (17). The models were subjected to 2000 steps of restrained conjugate gradient minimization in CNS followed by 1000 steps of unrestrained steepest descent minimization in CHARMm. All 3D graphical visualization and manipulation were accomplished with QUANTA 4.0, MIDAS (18) or MOLMOL (19). All helical
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FIG. 2. An expanded region of a NOESY spectrum (140 ms mixing time) in D 2O, pH 6.7 at 15°C showing selected drug-drug and drug-DNA connectivities. The crosspeaks are assigned as follows: (a) C4⬙Me-C3H1⬘; (b) C4⬙Me-H6⬙; (c) C4⬙Me-H2⬙; (d) C4⬘Me-H3⬙; (e) C4⬘Me-H5⬘b; (f) C15Me C3H1⬘; (g) C15Me-C3H5; (h) C15Me-H17; (i) C15Me-; (j) C2⬙Me-H2⬙; (k) C2⬙Me-H3⬙; (l) C2⬘Me-G10H1⬘; m) C2⬘Me-H6⬘; (n) C2⬘Me-G11H4⬘; (o) C2⬘Me-G11H5⬘; (p) C2⬘Me-H2⬘; (q) C2⬘Me-H3⬘; (r) C2⬘Me-H4⬘; (s) H6⬘-G10H1⬘; (t) H6⬘-H2⬘; (u) H6⬘-H3⬘; (v) H6⬙-G5H5⬘; (w) H6⬙-H5⬙b; (x) H6⬙-4⬙NMe.
parameters, torsion angles, and deoxyribose puckers were calculated with the aid of the program CURVES 4.1 (20).
RESULTS AND DISCUSSION Resonance Assignment of the Duplex Initial spectral assignments were made from 2D COSY, TOCSY and NOESY 1H spectra acquired at 400 MHz on a Varian Unity-400 and at 600 MHz on a Bruker DMX-600 spectrometer. The G4H8 resonance is absent from the spectra in D 2O buffer because of exchange with D 2O that results from the increase in acidity of H8 on adduct formation (6, 7). The H8 is observed at 9.65 ppm in NOESY spectra acquired in H 2O buffer, considerably downfield of its normal position. For the most part, the hedamycin resonances are close to those observed in similar complexes of heda-
mycin and oligonucleotides (6, 7). Figure 2 shows a selected region from a NOESY spectrum of the complex depicting some selected drug– drug and drug–DNA NOE contacts that are of particular importance for determining the F saccharide conformation and locating the position of the E and F saccharides in the minor groove. Structure Refinement and Analysis The quality of the final structures was assessed with the program CORMA (21) and the statistics are reported in Table 1. Comparison of the pairwise rmsd values between the ten structures of the ensemble and the average structure of the ensemble showed that the choice of A- or B-DNA as the starting conformation of d(ACCGGT) 2 had little effect on the final structure
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except at the terminal bases of d(ACCGGT) 2. In particular, the final hedamycin conformation was, within error, identical in both the A and the B ensembles.
TABLE 1
Structural Statistics and Root Mean Square Deviations for 10 NMR Structures of Hedamycin:d(ACCGGT) 2 Structural restraints Distance restraints Total Intraresidue Sequential (DNA) Drug–drug Drug–DNA Hydrogen bond restraints
392 176 54 106 40 16
Statistics for structure calculations Mean deviations from ideal covalent geometry Bond length (nm) Bond angle (°) Impropers (°) Atomic rmsds (nm) all residues (from mean) Standard R factors R intraresidue R interresidue R total Intramolecular CHARMM energies Total energy (kcal/mol) Electrostatic energy (kcal/mol)
0.005 1.7 1.07 0.056–0.083 0.51–0.54 0.51–0.59 0.52–0.55 ⫺709 ⫺1127
Oligonucleotide Conformation The relative intensities of the crosspeaks in this NOESY spectrum indicated that d(ACCGGT) 2 remains a B-form duplex in the presence of hedamycin. As reported previously (6, 7), there is an unbroken connectivity in the strand that does not contain the hedamycin adduct (strand 2), although the sequential C9H2⬘, C9H2⬘/G10H8 crosspeaks, which straddle the intercalation site, are weak. The opposite strand, containing the hedamycin adduct (strand 1), exhibits a broken connectivity pattern. The connectivity is broken at the C3-G4 step. All of the bases, including the modified G4 residue assume Watson–Crick alignments. The glycosidic torsion angle, the sugar pucker pseudorotations, the axial rise and helical twist are plotted in Fig. 3. The glycosidic torsion angles (Fig. 3A) for all bases are in the anti range and most of the deoxyribose sugars adopt pseudorotation angles in the range 160° (C2⬘ endo) to 110° (C1⬘ exo) (Fig. 3B). In the latter case, the only exceptions are the terminal resi-
FIG. 3. Helical parameters derived from the CURVES program for the hedamycin: (ACCGGT) 2 complex. The average value (open circles) and range (vertical bars) are displayed for (A) the glycosidic torsion angle, , (B) pseudorotation angle, P, (C) the axial rise at each base-pair step, and (D) the helical twist. The normal range of each of these parameters for A and B DNA are indicated along the right axis. 1605
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FIG. 4. Stereo stick model showing the stacking of the chromophore on the DNA bases bordering the intercalation site. The drug is displayed in black and the base pairs of d(ACCGGT) 2 on either side of hedamycin are displayed in gray.
dues A7 and T12 and one of the residues that borders the intercalation site, C3, which has P ⫽ 80°. The intercalation site is clearly seen in the graph of the axial rise versus base-pair step (Fig. 3C), being approximately 6.5 Å for the C3.G10-G4.C9 step, compared to an average rise in a 3– 4 Å range for all the other steps. All of the base-pair steps are unwound compared to Aand B-DNA except for the initial A1.T12-C2.G11 step (Fig. 3D). Inspection of the structure of the oligonucleotide in this complex reveals that the intercalation site is wedge-shaped with the widest part of the wedge at the 5⬘-C3-G4-3⬘ step and the narrowest part at the 5⬘-C9-G10-3⬘ step which is consistent with the nonexistent sequential NOEs at the former step and the weak sequential NOEs at the latter step. Conformation of the Epoxide Chain and the Interaction with the Oligonucleotide A large number of intermolecular NOEs between the duplex and hedamycin (42 NOE contacts) assist in aligning the drug with the duplex. NOEs between the duplex and the epoxide chain help locate the site of alkylation at G4 which agrees with the extreme lability of the H8 aromatic proton of G4 and its downfield position in the spectrum (6, 7). Similarly, large changes in the chemical shift of H18 and an intense NOE between the C19 methyl and G4H8 and moderate NOEs between the C15-methyl and H17 with G4H8 and a weak NOE between H18 and G4H8 confirm the site of covalent attachment at C18. Examination of the distances derived from MARDIGRAS analysis of the 80 ms mixing time NOESY crosspeak intensities show that the configuration of the epoxide chain is 14R, 16S, 17R, 18R as observed by Hansen et al. (7). Pavolopoulos et al. (6) reported a strong NOE between the C19methyl and the C5-methyl of a thymine on the 3⬘ side of the 5⬘-CG-3⬘ intercalation site. We observe no NOE contacts (Fig. 2) between the C19-methyl and the guanine base to the 3⬘ side of the 5⬘-CG-3⬘ site (i.e., G5). In fact, there appears to be no substantial contact be-
tween any of the hedamycin side-chain protons and G5 on the 3⬘ side of the intercalation site. Since the hedamycin adduct readily forms at this 5⬘-CGG-3⬘ binding site, the biological preference for 5⬘-CGT-3⬘ remains undetermined; it is difficult to resolve whether there is a conformational change within the 5⬘-CGG-3⬘ site in this study compared to the 5⬘-CGT-3⬘ of the previous work since no coordinates are publicly available for this study. Orientation of the Chromophore in the Intercalation Site The chromophore intercalates between the C3-G4/ G10-C9 base-pair step diagonally such that the 6-membered A ring of the chromophore is partially stacked over the 6-membered ring of G4, and the 6-membered C ring of the chromophore is partially stacked over the 6-membered ring of G10. A stereoview of the base stacking of the C3-G10 and G4-C9 base pairs (gray) flanking the hedamycin chromophore (black) is shown in Fig. 4. These stacking interactions obviously contribute to the stability of the complex but may also have a role in directing the epoxide ring to the alkylation site. The chromophore appears to be elegantly engineered to match the helical twist of the DNA duplex to maximize stacking interactions and orient the epoxide chain for optimal attack in the major groove at the N7 of the target guanine. Orientation of the Saccharide Rings The anglosamine (E) and N,N-dimethylvancosamine (F) saccharides both show NOEs exclusively to protons in the minor groove of the duplex. The two saccharide rings are oriented in the minor groove on either side of the intercalation site. The anglosamine (E) saccharide makes NOE contacts primarily with residues from the nonalkylated strand including G9 and G10. The N,Ndimethylvancosamine (F) saccharide has NOE contacts with residues on the alkylated strand including G4. It
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FIG. 5. Stereo stick model showing the orientation of the epoxide chain and the E and F saccharides relative to the chromophore and the DNA. Hedamycin is displayed in black and the four central base pairs of d(ACCGGT) 2 are displayed in gray.
is important to note that the C2⬙-methyl resonance of the F saccharide shows no NOE contacts to DNA resonances (Fig. 2) and hence must be oriented away from the DNA backbone toward the center of the duplex, in agreement with the model of Hansen et al. (7), but differing from the position of the F saccharide in the model of Pavolopoulos et al. (6) which has this C2⬙methyl located close to the DNA backbone. In contrast, the C2⬘-methyl resonance of the E saccharide has several NOE contacts with protons from the DNA backbone indicating a location near the minor groove wall. The plane of the E saccharide is approximately parallel to the minor groove wall but the plane of the F saccharide is tilted more toward the bottom of the minor groove in a orientation similar to the model of Hansen et al. (7) but different to the model of Pavolopoulos et al. (6), in which the F saccharide is very close to and parallel with the backbone of the DNA. In our model (Fig. 5), the F saccharide is neither close nor parallel to the minor groove wall. We find good agreement with the torsion angle of the E saccharide and the chromophore (i.e., C9-C8-C6⬘-C5⬘); ⫺101° in our model compared to ⫺100° in the model of Pavolopoulos et al. (6). However, the torsion angle for the F saccharide and the chromophore (i.e., C9-C10-C6⬙-C5⬙) of ⫺136° in our model differs from the 86° torsion angle in the model of Pavolopoulos et al. (6). The positively charged dimethylamino group in each saccharide is located near the O2 carbonyl groups of the two cytosines (C3 and C9) that flank the binding site. Neither of the dimethylamino groups of hedamycin and the carbonyl groups of C3 and C9 is sufficiently close for a direct hydrogen bond to form but it may be possible for water molecules to form a bridge between either of these dimethylamino groups and the carbonyl groups (7). The orientation of the F saccharide near the center of the minor groove calls into question the hypothesis that there is a higher degree of complementarity for hedamycin with the walls and the floor of the minor groove when the binding site is 5⬘-CG-3⬘ compared to 5⬘-TG-3⬘ (6). A more plausible mechanism is apparent
from the orientation of the C4⬙-methyl group of the F saccharide in the model presented in Figure 5. The C4⬙-methyl group is oriented toward the floor of the minor groove near the bulky guanine amino group of G10. If the binding site was 5⬘-TG-3⬘, then this base would be an adenine and the bulky amino group is replaced by a proton. The steric interaction between the methyl group of the F saccharide and the bulky amino group of guanine has the effect of pushing the F saccharide closer to the wall of the minor groove and creating a more effective interaction between the positively charged dimethylamino group and the carbonyl group of cytosine either by direct interaction or by a water mediated hydrogen bond. In summary, we have calculated a high resolution NMR structure of hedamycin bound to an oligonucleotide with a 5⬘-CGG-3⬘ binding site. The structure shares many common features of two previous NMR structures of hedamycin:DNA complexes but differs in the details of the conformation and orientation of the N,N-dimethylvancosamine saccharide in the minor groove with the structure that has the same 5⬘-CGG-3⬘ binding site. We have deduced a correlation between the N,N-dimethylvancosamine substitution at the C10 position on the chromophore and the preference for 5⬘-CG-3⬘ over 5⬘-TG-3⬘ binding sites but there is no conclusive evidence from the structure for the preference for 5⬘-CGT-3⬘ binding sites over 5⬘-CGG-3⬘ binding sites. ACKNOWLEDGMENTS This work has benefited from the use of NMR facilities at the Australian National University. The authors are grateful to Professor Tom James, University of California, San Francisco for a copy of the programs, CORMA and MARDIGRAS, Professor Richard Lavery, Institut de Biologie Physico-Chimique, Paris for a copy of the program, CURVES 4.1, Professor Kurt Wu¨thrich, ETH Zu¨rich for a copy of the programs XEASY and MOLMOL, and Professor Axel Bru¨nger, Stanford University for a copy of the program CNS.
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Structural Investigation of the Hedamycin:d(ACCGGT) 2 Complex by NMR and Restrained Molecular Dynamics Elisabeth A. Owen,* Glenn A. Burley,† John A. Carver,† Geoffrey Wickham,† ,1 and Max A. Keniry* ,2 *Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia; and †Department of Chemistry, University of Wollongong, Wollongong, New South Wales 2522, Australia
Received November 26, 2001
Hedamycin, a member of the pluramycin family of drugs, displays a range of biological responses including antitumor and antimicrobial activity. The mechanism of action is via direct interaction with DNA through intercalation between the bases of the oligonucleotide and alkylation of a guanine residue at 5ⴕPyG-3ⴕ sites. There appears to be some minor structural differences between two earlier studies on the interaction of hedamycin with 5ⴕ-PyG-3ⴕ sites. In this study, a high-resolution NMR analysis of the hedamycin:d(ACCGGT) 2 complex was undertaken in order to investigate the effect of replacing the thymine with a guanine at the preferred 5ⴕ-CGT-3ⴕ site. The resultant structure was compared with earlier work, with particular emphasis placed on the drug conformation. The structure of the hedamycin:d(ACCGGT) 2 complex has many features in common with the two previous NMR structures of hedamycin:DNA complexes but differed in the conformation and orientation of the N,Ndimethylvancosamine saccharide of hedamycin in one of these structures. The preferential binding of hedamycin to 5ⴕ-CG-3ⴕ over 5ⴕ-TG-3ⴕ binding sites is explained in terms of the orientation and location of the N,N-dimethylvancosamine saccharide in the minor groove. © 2002 Elsevier Science (USA) Key Words: 2D NMR; drug–DNA; hedamycin; oligonucleotide.
Coordinates: The coordinates of the hedamycin:d(ACCGGT) 2 complex have been deposited with the Protein Data Bank (Accession No. 1JHI). Abbreviations used: NMR, nuclear magnetic resonance; COSY, correlation spectroscopy; NOESY, two-dimensional nuclear Overhauser exchange spectroscopy; PCOSY, purged correlation spectroscopy; TOCSY, total correlation spectroscopy. 1 Present address: Polymerat Pty. Ltd., St Lucia, Queensland 4067, Australia. 2 To whom correspondence and reprint requests should be addressed. Fax: ⫹61 2 6125 0750. E-mail: [email protected]. 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
Hedamycin (Fig. 1) is a naturally occurring antibiotic from the pluramycin family of antibiotics derived from the Streptomyces genera of fungi. Interest in the pluramycins arose from a range of biological responses, such as antitumor and antimicrobial activity (1, 2). Structurally, hedamycin consists of a highly substituted 4H-anthra[1,2-b]-pyran-4,7,12-trione chromophore with two amino sugars, an anglosamine attached to the C8 position and an N,N-dimethyvancosamine attached to the C10 position (Fig. 1) (1). Hedamycin alkylates nucleic acids at guanine sites with high 5⬘-PyGT-3⬘ selectivity but favors 5⬘-CGT-3⬘ sequences (3–5). Substantial adduct formation is also observed at 5⬘-CGG-3⬘ sequences (4). Previous NMR studies have shown that the anthrapyrantrione chromophore of hedamycin intercalates between the 5⬘PyG-3⬘ bases with the two amino sugars placed in the minor groove and the bisepoxide chain located in the major groove (5–7). The anglosamine sugar is located to the 3⬘ side of the intercalation site and the N,Ndimethylvancosamine sugar is located to the 5⬘ side of the intercalation site. The preferential selectivity for a pyrimidine on the 5⬘ side of the alkylated guanine appears to arise from an interaction between the O2 carbonyl of the pyrimidine and the positively charged dimethylamino substituent of the N,N-dimethylvancosamine sugar (6). The preference for cytosine over thymine in this position is less obvious although it has been suggested that more extensive van der Waals interactions between the sugar and the wall of the minor groove favor the cytosine base (6). Here we present an NMR-derived, solution phase, three dimensional model of the hedamycin:d(ACCGGT) 2 complex that was determined by relaxation matrix refinement of the NOESY intensities, followed by torsion angle molecular dynamics and energy minimization. The model of hedamycin:d(ACCGGT) 2 is in substantial agreement with previous NMR studies of hedamycin–DNA complexes (6, 7) but differs in detail with one of these studies (6). The model calls into
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 600 NMR spectrometers using COSY, TOCSY and NOESY spectra. A heteronuclear proton-detected 31P– 1H correlation experiment was collected at 600 MHz on a Varian INOVA 600 spectrometer by the g-HELCO technique (10). Quantitative two-dimensional NOESY spectra were acquired on a Varian INOVA 600 spectrometer. These data included NOESY spectra (80, 140, and 200 ms mixing times, 3.5 s recycle time) of the ⬃1 mM hedamycin-d(ACCGGT) 2 complex in 10 mM phosphate, 50 mM NaCl, in pH 6.7 D 2O buffer at 15°C and a NOESY spectrum (250 ms mixing time) of the complex in a similar 90% H 2O/10% D 2O buffer at 15°C. All spectra were processed with Varian VNMR software (Varian Associates, Palo Alto) and quantitated and visualized using XEASY software (11). Typically 4096 complex points were collected over 5200 Hz in the t 2 domain and 700 complex points over 5200 Hz in the t 1 domain. Datasets were zerofilled to a final size of 4096 ⫻ 2048 complex points prior to Fourier transformation.
FIG. 1. Structure of hedamycin and the DNA sequence showing the intercalation site. The modified guanine is shown in bold.
question the mechanism by which cytosine is favored over thymine at the 5⬘ side of the 5⬘-PyG-3⬘ intercalation site (6). We propose a different mechanism for this selectivity that is consistent with the evolution of a N,N-dimethylvancosamine sugar at the C10 position on the chromophore. Hedamycin clearly interacts with the 5⬘-CGG-3⬘ site by covalent adduct formation at the central guanine position but there appears to be no obvious reason for a preference for thymine at the 3⬘ position in this sequence as indicated by sequence selectivity studies (8). MATERIALS AND METHODS Sample preparation. The hedamycin used in this work was a generous gift from Bristol-Myers Squibb and was used without further purification. d(ACCGGT) was synthesized on an Applied Biosystems Model 391A DNA synthesizer using solid phase phosphoramidite chemistry and purified by reverse phase HPLC followed by extensive dialysis. The drug–DNA adduct was formed by adding 10 l aliquots of a 10 mM solution of hedamycin to a 1.0 mM solution of d(ACCGGT) 2 in 10 mM phosphate, 50 mM NaCl, pH 6.7 D 2O buffer. The titrations were conducted in the dark and nitrogen was bubbled through the reaction mixture after the addition of each drug aliquot to prevent photo-oxidation of the ligand (9). The mixture was characterized after each addition by the acquisition of 1H NMR spectra. After the final addition of hedamycin at a 1:1 ratio of hedamycin: d(ACCGGT) 2 the complex was allowed to stand for 12 h at 25°C to ensure complete reaction. NMR spectroscopy. Initial characterization and assignment of the complex was performed on Varian Unity-400 and Bruker DMX-
Experimental restraints. Initially, crosspeaks between the nonexchangeable protons were divided into strong, medium and weak categories and given bounds of 0.2– 0.35, 0.35– 0.45 and 0.4 – 0.55 nm, respectively. A similar strategy was adopted for the crosspeaks that involved exchangeable protons in the NOESY spectra of the complex in 90% H 2O/10% D 2O buffer. In addition, 16 restraints were created to maintain the hydrogen bonds between the bases of the two nucleotide chains and given the bonds: GC pairs, 0.285– 0.305 nm (G-O6 to C-N4), (G-N1 to C-N3) and 0.276 – 0.296 nm (G-N2 to C-O2); AT pairs, 0.275– 0.295 nm (A-N1 to T-N3) and 0.285– 0.205 nm (A-N6 to T-O4). Backbone dihedral angles were conservatively restrained about 0° ⫾ 150° (␣), 180° ⫾ 60° (), 60° ⫾ 60° (␥) and 0° ⫾ 150° () following analysis of 31P chemical shifts (12), H2⬘-H5⬘/H5⬙ and H6/ H8-H5⬘/H5⬙ NOEs and H3⬘, H4⬘ and H5⬘/H5⬙ linewidths in the NOESY spectra following the procedure of Reid et al. (13, 14). Structure determination and refinement. Interproton distance restraints were estimated from the crosspeak intensity of the 2D NOESY spectra by employing the MARDIGRAS (Version 5.2) program and the pseudoenergy force field was derived from these restraints as described in Keniry et al. (15). The initial model of self-complementary d(ACCGGT) 2 was created from standard A- and B-conformation DNA coordinates using the QUANTA 4.0 Nucleic Acid Builder (MSI, Waltham, MA). The hedamycin structure was generated from the crystal structure coordinates (16). The complex was formed by breaking the epoxide ring containing the C18 carbon and attaching C18 covalently to the N7 of G4 in the sequence d(A1C2-C3-G4-G5-T6). The chromophore was placed between the C3-G10 and G4-C9 base pairs and the two saccharides located on the minor groove side of the oligonucleotide. The initial models were extensively minimized and then subjected to Cartesian space molecular dynamics using the QUANTA/ CHARMm 23.1 package (MSI, Waltham, MA) on a Silicon Graphics O2 workstation. The models were further refined using torsion angle dynamics protocol of CNS (17) against distances derived from crosspeak intensities obtained from NOESY spectra with mixing times of 80, 150, and 200 ms and refined with several iterations of MARDIGRAS. Crosspeak intensities for the exchangeable protons were not subject to relaxation matrix analysis but instead were assigned distance bounds as described above. In all, there were 394 distance restraints. Of these, 307 were estimated from the NOESY volumes and 64 other restraints derived from the NMR spectra that were converted into distance restraints. A version of the standard anneal protocol of CNS, modified to allow up to 60 ps of torsion angle dynamics at 20,000°K was used. The models were cooled from 20,000 to 1,000°K over 76 cycles of 50 steps (each cycle taking 0.75 ps). Final cooling from 1,000 to 300°K was over 70 cycles of 30 steps using Cartesian dynamics (17). The models were subjected to 2000 steps of restrained conjugate gradient minimization in CNS followed by 1000 steps of unrestrained steepest descent minimization in CHARMm. All 3D graphical visualization and manipulation were accomplished with QUANTA 4.0, MIDAS (18) or MOLMOL (19). All helical
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FIG. 2. An expanded region of a NOESY spectrum (140 ms mixing time) in D 2O, pH 6.7 at 15°C showing selected drug-drug and drug-DNA connectivities. The crosspeaks are assigned as follows: (a) C4⬙Me-C3H1⬘; (b) C4⬙Me-H6⬙; (c) C4⬙Me-H2⬙; (d) C4⬘Me-H3⬙; (e) C4⬘Me-H5⬘b; (f) C15Me C3H1⬘; (g) C15Me-C3H5; (h) C15Me-H17; (i) C15Me-; (j) C2⬙Me-H2⬙; (k) C2⬙Me-H3⬙; (l) C2⬘Me-G10H1⬘; m) C2⬘Me-H6⬘; (n) C2⬘Me-G11H4⬘; (o) C2⬘Me-G11H5⬘; (p) C2⬘Me-H2⬘; (q) C2⬘Me-H3⬘; (r) C2⬘Me-H4⬘; (s) H6⬘-G10H1⬘; (t) H6⬘-H2⬘; (u) H6⬘-H3⬘; (v) H6⬙-G5H5⬘; (w) H6⬙-H5⬙b; (x) H6⬙-4⬙NMe.
parameters, torsion angles, and deoxyribose puckers were calculated with the aid of the program CURVES 4.1 (20).
RESULTS AND DISCUSSION Resonance Assignment of the Duplex Initial spectral assignments were made from 2D COSY, TOCSY and NOESY 1H spectra acquired at 400 MHz on a Varian Unity-400 and at 600 MHz on a Bruker DMX-600 spectrometer. The G4H8 resonance is absent from the spectra in D 2O buffer because of exchange with D 2O that results from the increase in acidity of H8 on adduct formation (6, 7). The H8 is observed at 9.65 ppm in NOESY spectra acquired in H 2O buffer, considerably downfield of its normal position. For the most part, the hedamycin resonances are close to those observed in similar complexes of heda-
mycin and oligonucleotides (6, 7). Figure 2 shows a selected region from a NOESY spectrum of the complex depicting some selected drug– drug and drug–DNA NOE contacts that are of particular importance for determining the F saccharide conformation and locating the position of the E and F saccharides in the minor groove. Structure Refinement and Analysis The quality of the final structures was assessed with the program CORMA (21) and the statistics are reported in Table 1. Comparison of the pairwise rmsd values between the ten structures of the ensemble and the average structure of the ensemble showed that the choice of A- or B-DNA as the starting conformation of d(ACCGGT) 2 had little effect on the final structure
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except at the terminal bases of d(ACCGGT) 2. In particular, the final hedamycin conformation was, within error, identical in both the A and the B ensembles.
TABLE 1
Structural Statistics and Root Mean Square Deviations for 10 NMR Structures of Hedamycin:d(ACCGGT) 2 Structural restraints Distance restraints Total Intraresidue Sequential (DNA) Drug–drug Drug–DNA Hydrogen bond restraints
392 176 54 106 40 16
Statistics for structure calculations Mean deviations from ideal covalent geometry Bond length (nm) Bond angle (°) Impropers (°) Atomic rmsds (nm) all residues (from mean) Standard R factors R intraresidue R interresidue R total Intramolecular CHARMM energies Total energy (kcal/mol) Electrostatic energy (kcal/mol)
0.005 1.7 1.07 0.056–0.083 0.51–0.54 0.51–0.59 0.52–0.55 ⫺709 ⫺1127
Oligonucleotide Conformation The relative intensities of the crosspeaks in this NOESY spectrum indicated that d(ACCGGT) 2 remains a B-form duplex in the presence of hedamycin. As reported previously (6, 7), there is an unbroken connectivity in the strand that does not contain the hedamycin adduct (strand 2), although the sequential C9H2⬘, C9H2⬘/G10H8 crosspeaks, which straddle the intercalation site, are weak. The opposite strand, containing the hedamycin adduct (strand 1), exhibits a broken connectivity pattern. The connectivity is broken at the C3-G4 step. All of the bases, including the modified G4 residue assume Watson–Crick alignments. The glycosidic torsion angle, the sugar pucker pseudorotations, the axial rise and helical twist are plotted in Fig. 3. The glycosidic torsion angles (Fig. 3A) for all bases are in the anti range and most of the deoxyribose sugars adopt pseudorotation angles in the range 160° (C2⬘ endo) to 110° (C1⬘ exo) (Fig. 3B). In the latter case, the only exceptions are the terminal resi-
FIG. 3. Helical parameters derived from the CURVES program for the hedamycin: (ACCGGT) 2 complex. The average value (open circles) and range (vertical bars) are displayed for (A) the glycosidic torsion angle, , (B) pseudorotation angle, P, (C) the axial rise at each base-pair step, and (D) the helical twist. The normal range of each of these parameters for A and B DNA are indicated along the right axis. 1605
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FIG. 4. Stereo stick model showing the stacking of the chromophore on the DNA bases bordering the intercalation site. The drug is displayed in black and the base pairs of d(ACCGGT) 2 on either side of hedamycin are displayed in gray.
dues A7 and T12 and one of the residues that borders the intercalation site, C3, which has P ⫽ 80°. The intercalation site is clearly seen in the graph of the axial rise versus base-pair step (Fig. 3C), being approximately 6.5 Å for the C3.G10-G4.C9 step, compared to an average rise in a 3– 4 Å range for all the other steps. All of the base-pair steps are unwound compared to Aand B-DNA except for the initial A1.T12-C2.G11 step (Fig. 3D). Inspection of the structure of the oligonucleotide in this complex reveals that the intercalation site is wedge-shaped with the widest part of the wedge at the 5⬘-C3-G4-3⬘ step and the narrowest part at the 5⬘-C9-G10-3⬘ step which is consistent with the nonexistent sequential NOEs at the former step and the weak sequential NOEs at the latter step. Conformation of the Epoxide Chain and the Interaction with the Oligonucleotide A large number of intermolecular NOEs between the duplex and hedamycin (42 NOE contacts) assist in aligning the drug with the duplex. NOEs between the duplex and the epoxide chain help locate the site of alkylation at G4 which agrees with the extreme lability of the H8 aromatic proton of G4 and its downfield position in the spectrum (6, 7). Similarly, large changes in the chemical shift of H18 and an intense NOE between the C19 methyl and G4H8 and moderate NOEs between the C15-methyl and H17 with G4H8 and a weak NOE between H18 and G4H8 confirm the site of covalent attachment at C18. Examination of the distances derived from MARDIGRAS analysis of the 80 ms mixing time NOESY crosspeak intensities show that the configuration of the epoxide chain is 14R, 16S, 17R, 18R as observed by Hansen et al. (7). Pavolopoulos et al. (6) reported a strong NOE between the C19methyl and the C5-methyl of a thymine on the 3⬘ side of the 5⬘-CG-3⬘ intercalation site. We observe no NOE contacts (Fig. 2) between the C19-methyl and the guanine base to the 3⬘ side of the 5⬘-CG-3⬘ site (i.e., G5). In fact, there appears to be no substantial contact be-
tween any of the hedamycin side-chain protons and G5 on the 3⬘ side of the intercalation site. Since the hedamycin adduct readily forms at this 5⬘-CGG-3⬘ binding site, the biological preference for 5⬘-CGT-3⬘ remains undetermined; it is difficult to resolve whether there is a conformational change within the 5⬘-CGG-3⬘ site in this study compared to the 5⬘-CGT-3⬘ of the previous work since no coordinates are publicly available for this study. Orientation of the Chromophore in the Intercalation Site The chromophore intercalates between the C3-G4/ G10-C9 base-pair step diagonally such that the 6-membered A ring of the chromophore is partially stacked over the 6-membered ring of G4, and the 6-membered C ring of the chromophore is partially stacked over the 6-membered ring of G10. A stereoview of the base stacking of the C3-G10 and G4-C9 base pairs (gray) flanking the hedamycin chromophore (black) is shown in Fig. 4. These stacking interactions obviously contribute to the stability of the complex but may also have a role in directing the epoxide ring to the alkylation site. The chromophore appears to be elegantly engineered to match the helical twist of the DNA duplex to maximize stacking interactions and orient the epoxide chain for optimal attack in the major groove at the N7 of the target guanine. Orientation of the Saccharide Rings The anglosamine (E) and N,N-dimethylvancosamine (F) saccharides both show NOEs exclusively to protons in the minor groove of the duplex. The two saccharide rings are oriented in the minor groove on either side of the intercalation site. The anglosamine (E) saccharide makes NOE contacts primarily with residues from the nonalkylated strand including G9 and G10. The N,Ndimethylvancosamine (F) saccharide has NOE contacts with residues on the alkylated strand including G4. It
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FIG. 5. Stereo stick model showing the orientation of the epoxide chain and the E and F saccharides relative to the chromophore and the DNA. Hedamycin is displayed in black and the four central base pairs of d(ACCGGT) 2 are displayed in gray.
is important to note that the C2⬙-methyl resonance of the F saccharide shows no NOE contacts to DNA resonances (Fig. 2) and hence must be oriented away from the DNA backbone toward the center of the duplex, in agreement with the model of Hansen et al. (7), but differing from the position of the F saccharide in the model of Pavolopoulos et al. (6) which has this C2⬙methyl located close to the DNA backbone. In contrast, the C2⬘-methyl resonance of the E saccharide has several NOE contacts with protons from the DNA backbone indicating a location near the minor groove wall. The plane of the E saccharide is approximately parallel to the minor groove wall but the plane of the F saccharide is tilted more toward the bottom of the minor groove in a orientation similar to the model of Hansen et al. (7) but different to the model of Pavolopoulos et al. (6), in which the F saccharide is very close to and parallel with the backbone of the DNA. In our model (Fig. 5), the F saccharide is neither close nor parallel to the minor groove wall. We find good agreement with the torsion angle of the E saccharide and the chromophore (i.e., C9-C8-C6⬘-C5⬘); ⫺101° in our model compared to ⫺100° in the model of Pavolopoulos et al. (6). However, the torsion angle for the F saccharide and the chromophore (i.e., C9-C10-C6⬙-C5⬙) of ⫺136° in our model differs from the 86° torsion angle in the model of Pavolopoulos et al. (6). The positively charged dimethylamino group in each saccharide is located near the O2 carbonyl groups of the two cytosines (C3 and C9) that flank the binding site. Neither of the dimethylamino groups of hedamycin and the carbonyl groups of C3 and C9 is sufficiently close for a direct hydrogen bond to form but it may be possible for water molecules to form a bridge between either of these dimethylamino groups and the carbonyl groups (7). The orientation of the F saccharide near the center of the minor groove calls into question the hypothesis that there is a higher degree of complementarity for hedamycin with the walls and the floor of the minor groove when the binding site is 5⬘-CG-3⬘ compared to 5⬘-TG-3⬘ (6). A more plausible mechanism is apparent
from the orientation of the C4⬙-methyl group of the F saccharide in the model presented in Figure 5. The C4⬙-methyl group is oriented toward the floor of the minor groove near the bulky guanine amino group of G10. If the binding site was 5⬘-TG-3⬘, then this base would be an adenine and the bulky amino group is replaced by a proton. The steric interaction between the methyl group of the F saccharide and the bulky amino group of guanine has the effect of pushing the F saccharide closer to the wall of the minor groove and creating a more effective interaction between the positively charged dimethylamino group and the carbonyl group of cytosine either by direct interaction or by a water mediated hydrogen bond. In summary, we have calculated a high resolution NMR structure of hedamycin bound to an oligonucleotide with a 5⬘-CGG-3⬘ binding site. The structure shares many common features of two previous NMR structures of hedamycin:DNA complexes but differs in the details of the conformation and orientation of the N,N-dimethylvancosamine saccharide in the minor groove with the structure that has the same 5⬘-CGG-3⬘ binding site. We have deduced a correlation between the N,N-dimethylvancosamine substitution at the C10 position on the chromophore and the preference for 5⬘-CG-3⬘ over 5⬘-TG-3⬘ binding sites but there is no conclusive evidence from the structure for the preference for 5⬘-CGT-3⬘ binding sites over 5⬘-CGG-3⬘ binding sites. ACKNOWLEDGMENTS This work has benefited from the use of NMR facilities at the Australian National University. The authors are grateful to Professor Tom James, University of California, San Francisco for a copy of the programs, CORMA and MARDIGRAS, Professor Richard Lavery, Institut de Biologie Physico-Chimique, Paris for a copy of the program, CURVES 4.1, Professor Kurt Wu¨thrich, ETH Zu¨rich for a copy of the programs XEASY and MOLMOL, and Professor Axel Bru¨nger, Stanford University for a copy of the program CNS.
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