ABSTRACT
The crystal structure is reported of a complex between the dodecanucleotide sequence d(CGCGAATTCGCG)2 and an analogue of the DNA binding drug Hoechst 33258, in which the piperazine ring has been replaced by an amidinium group and the phenol ring by a phenylamidinium group. The structure has been refined to an R factor of 19.5% at 2.2 Å resolution. The drug is held in the minor groove by five strong hydrogen bonds, together with bridging water molecules at both ends. There are few other contacts with the floor of the groove, indicating a lack of isohelicity with the groove and suggesting (i) that the observed high DNA affinity of this drug is primarily due to the array of hydrogen bonds and (ii) that these more than compensate for its poor isohelicity.
The minor groove of DNA is the locus of action of a large number of drugs, typified by the antiviral agents netropsin and distamycin, the anti-Pneumocystis carinii drug pentamidine, the trypanocidal drug berenil and the experimental antitumour agent and DNA stain Hoechst 33258. These molecules interact in AT regions of B-form duplex DNA (1 -3 ), where they can act as blocks to transcription (4 ) or to the action of DNA topoisomerase enzymes (5 ,6 ).
The interactions of these molecules with DNA have been studied by a range of biophysical and structural techniques (1 -3 ). Spectroscopic and footprinting methods (see for example 7 -9 ) have concurred with numerous X-ray crystallographic studies (10 -25 ) on drug-oligonucleotide complexes in defining the binding sites and their size and have also helped to illuminate the nature of the sequence selectivities shown by these drugs at the molecular level. The relative contributions of various factors to these sequence selectivities continues to be debated, not least in view of their importance for the design of new agents, including those with altered selectivities. The major factors (26 ,27 ) in minor (and major) groove sequence preferences are: (i) hydrogen bonding from drug to base pair edges; (ii) electrostatic interactions between, in particular, the cationic regions of the drugs and the negative electrostatic potential of AT-rich minor grooves; (iii) non-bonded van der Waals interactions with the floor and/or walls of the groove; (iv) specific water/drug/DNA hydrogen bonding; (v) intrinsic sequence-dependent features of DNA structure at a particular locus, especially groove width and base morphology. The shape of the drug molecule itself can also play a role, with a requirement for shape isohelical with the curvature of the groove being highlighted by several studies (28 -30 ).
We have used cationic benzimidazoles (Fig. 1 ) as experimental probes of minor groove recognition (31 ,32 ) in which the nature of the cationic group, the number of charges and the number of benzimidazole groups has been varied. We have shown (31 ) that molecules with two benzimidazole groups produce a significantly greater increase in melting temperature ([Delta]Tm) with the dodecanucleotide d(CGCGAATTCGCG)2, of 16-18oC (a free energy difference of ~3 kcal/mol; 31 ), compared with molecules having just one benzimidazole group and with the same charged end group (such as an imidazole ring or amidinium group), with [Delta]Tm values of 8-10oC (a free energy difference of ~2 kcal/mol; 31 ). This suggests that van der Waals contacts are at least as important as electrostatic interactions for overall minor groove binding. Binding of the groove binder netropsin is enthalpy driven (33 ), suggesting the same for the similar sized molecules discussed here. The crystal structure of compound 1 complexed with d(CGCGAATTCGCG)2 shows (22 ,31 ) that this ligand has fewer hydrogen bonds to the DNA than compound 2 (Hoechst 33258), yet more non-bonded contacts (as well as a significantly higher [Delta]Tm value, by 6oC). The novel compound 3 (DIBIZ), the subject of the present study, has a [Delta]Tm some 15oC higher than Hoechst 33258 itself and 9oC higher than compound 1. Compound 3 incorporates both the bis-benzimidazole moiety of Hoechst 33258 and the amidinium end groups present in berenil (12 ,14 ) and pentamidine (13 ), which tend to hydrogen bond to base pair edges. The present study reports on the crystal structure of compound 3 complexed with d(CGCGAATTCGCG)2 and examines its enhanced DNA affinity in structural terms, in order to obtain further insight into the determinants of its minor groove binding.
The DNA dodecamer d(CGCGAATTCGCG)2 was purchased from the Oswel DNA Service (University of Edinburgh) and annealed before use. The DIBIZ ligand was used as the hydrochloride salt. The synthesis of DIBIZ has been reported elsewhere (34 ).
The complex was grown from hanging drops at 286 K as colourless polyhedral crystals. The crystal used for data collection was grown from a drop containing 2 [mu]l 25% 2-methylpentane-2,4-diol, 2 [mu]l 5 mM DIBIZ, 2 [mu]l 15 mM MgCl2, 2 [mu]l 2.5 mM spermine and 2 [mu]l 5 mM dodecamer equilibrated against a reservoir containing 1 ml 40% 2-methyl-2,4-pentanediol. The DNA solution was prepared using 30 mM sodium cacodylate buffer, pH 7.0. The crystal employed for the X-ray study was obtained after 2 months.
The crystal used was of approximate dimensions 0.4 * 0.2 * 0.2 mm and was mounted inside a 0.5 mm Lindemann glass capillary with a small amount of mother liquor. Intensity data were collected at 15oC using a Siemens-Xentronics multiwire area detector with a rotating anode X-ray generator (40 mA, 75 kV) and a graphite monochromator. A crystal-detector distance of 10 cm and swing angle of 15o was used to collect data to a maximum possible resolution of 2.2 Å. Data were collected with [chi] set at 45o, while the crystal was rotated through 100o in [omega] at [Phi] values of 0o and 60o; 180 s frames were recorded every 0.20o step. The crystal did not suffer any observable decay during data collection. Data processing was carried out using the program package XENGEN version 1.3. After merging, the data comprised 3315 of the possible 3740 unique reflections to 2.2 Å (89%) with a merging R value of 4.4%.
The unit cell dimensions of the crystal are a = 25.75, b = 40.56, c = 66.26 Å, in the orthorhombic space group P212121. This cell is close to that of the native d(CGCGAATTCGCG)2 dodecamer (35 ) (a = 24.87, b = 40.39, c = 66.20 Å) and other groove-bound dodecamer-drug complexes (10 -25 ), suggesting that the present crystal structure is isomorphous with them. The coordinates used as a starting model for the structure refinement were those of the d(CGCGAATTCGCG)2 dodecamer. Crystallographic refinement was carried out using the program X-PLOR, version 3.1 (36 ). Rigid body refinement of the DNA molecule as one constrained group was performed with the resolution range of the data increased from 8.0-4.0 (568 reflections) to 8.0-3.5 Å (880 reflections). The R value was 35.3% at this point. The molecule was then divided into 24 rigid groups comprising individual nucleotides. The resolution range was gradually increased from 8.0-4.0 to 8.0-3.0 Å (1416 reflections), the R factor then being 27.7%. Individual positional refinement ultimately included all data to 2.2 Å (2954 reflections) and gave an R factor of 30.3%. Temperature factor refinement reduced the R value to 28.6%. Electron density maps were calculated and displayed using the graphics package TOM/FRODO, version 3.2.
The DNA molecule fitted the density well and a long continuous lobe of density was clearly visible in the minor groove. This lobe displayed sufficient structure to enable the correct orientation and positioning of the drug molecule to be deduced at the outset. Particular features were the two characteristic bulges for the benzimidazole groups and the thin aromatic ring sitting vertically in the groove at one end (Fig. 2 ). The density contained a longer region for the aromatic ring and its co-linear benzimidazole and a bend to a shorter region for the second benzimidazole. A structure for the DIBIZ molecule was constructed by means of molecular modelling (Hyperchem, version 4; Hypercube Inc.). The step-wise twist of the benzimidazoles observed in other complexes (~19o per benzimidazole) was incorporated in the model, which then closely fitted the electron density. At no time was there any indication of mobility of the drug along the minor groove. Electrostatic charges for the DIBIZ molecule were calculated with Hyperchem using modified neglect of differential overlap (MNDO) wave functions and the force field parameters were interpolated from previous studies in this laboratory. Planar restraints were applied to each individual aromatic ring system.
The structure of the complex between DIBIZ and the DNA dodecamer d(CGCGAATTCGCG)2 is shown in Figure 3 . The overall structure of the DNA duplex itself is consistent with other isomorphous dodecamers and dodecamer-drug complexes. The helix is right handed DNA. There are 9.8 bp per turn of the helix, with a mean helical twist angle between base pairs of 36o. The mean rise per base pair is 3.37 Å. The crystallographic asymmetric unit consists of two chemically equivalent self-complementary dodecanucleotide strands in an antiparallel duplex. The drug is bound in the 5'-AATT region of the minor groove of the duplex and occupies a single position with no evidence of orientational disorder or mobility along the groove. The molecule is twisted at each of its subunits in order to follow the helical curvature of the minor groove. This twist is in addition to the natural crescent shape of the molecule, as seen in Figure 1 . Dihedral angles between the aromatic rings of the two benzimidazole moieties are 20o and the terminal benzene ring is twisted by a further 20o out of the plane of its attached benzimidazole moiety.
Table 2
The DIBIZ molecule makes five direct hydrogen bonds with base pair edges (Table 2 and Figs 3 -5 ). There are two sets of interstrand bifurcated, three-centre hydrogen bonds, one involving the inner facing benzimidazole nitrogen atom N3 and the other the inner facing amidinium nitrogen atom N1, belonging to the same benzimidazole ring. This pattern of hydrogen bonding to O2 of thymines and N3 of adenines in the floor of the minor groove is typical of benzimidazole-containing drugs of the Hoechst 33258 family (15 ,17 -22 ,24 ). The observation of two hydrogen bonds from the terminal amidinium nitrogen N3 is less expected; in the context of drugs such as berenil, pentamidine and their analogues, invariably only a sole hydrogen bond is made from an amidinium to a base edge (8 ,12 -14 ,16 ,23 ,25 ). In the case of DIBIZ, atom N3 corresponds to the nitrogen of a third benzimidazole ring (Fig. 6 ) and thus the hydrogen bonding characteristics of the molecule resemble those observed in a tris(benzimidazole) complex (24 ). There are no hydrogen bonds between bases and the amidinium group at the 5'-end of the duplex.
Binding of the DIBIZ molecule in the minor groove is stabilized by van der Waals interactions between its benzimidazole and phenyl groups and (i) the sugar-phosphate backbone on the walls of the groove and (ii) the floor of the groove. There are a number of close contacts in the first category, primarily involving atoms O4', C4' and C5' from the DNA. In contrast, there are remarkably few contacts between drug and groove floor, apart from the hydrogen bonds detailed above. The only two of consequence are: (i) atom C18 on the inner face of the 5'-end phenyl ring is 3.4 Å away from N3A17; (ii) atom C3 on the 3' benzimidazole ring is 3.9 Å away from C2A6.
The groove width is inherently narrow in the AT-rich region of dodecamer duplexes (26 ,34 ). Groove widths (Fig. 7 ) indicate that the benzimidazole ligands produce a slight widening of the groove compared with the native structure. This is to be expected in view of the non-zero dihedral angles between the subunits in these drugs, which have the effect of forcing the two DNA strands somewhat apart. The complexes of DIBIZ and compound 2 produce very similar groove widths, which reflects their similar patterns of base pair propeller twist.
The DIBIZ molecule is unusual among minor groove binders. Its lack of a significant concave inner surface (see Figs 1 and 2 ) means that its shape does not conform to the general pattern of isohelicity (28 -30 ) observed in drugs such as netropsin, berenil, Hoechst 33258 and pentamidine. This is reflected in the present crystal structure, with very few van der Waals contacts between drug and groove floor, in striking contrast to these other drugs. Yet solution studies unequivocally indicate that DIBIZ is a strong DNA binder. The structure presented here suggests that the unusually large number of direct drug-DNA hydrogen bonds, together with well-formed water-drug-DNA bridges, is a major factor contributing to its binding. The structural results also suggest that hydrogen bonding, together with van der Waals interactions with the groove walls and electrostatic interactions, may balance out a strict requirement for isohelicity with the minor groove floor. Such considerations may be of use when designing further molecules for minor groove sequence recognition. The existence of A/T-selective molecules which cannot hydrogen bond to base pair edges, such as the bis-quaternary family of extended phenylamides (39 ,40 ), shows that the ability to form such hydrogen bonds is not an absolute requirement for groove binding.
This work was supported by CRC programme grant SP1384 (to S.N.) and NIH grant 33363 (to D.W.B.). G.R.C. is grateful to the University of Auckland for sabbatical leave at The Institute of Cancer Research during which this work was performed. We are grateful to Christine Nunn, John Trent and Alexis Wood (ICR) for discussions and assistance on crystallographic aspects of this work.
*To whom correspondence should be addressed. Tel/Fax: +44 181 643 1675; Email: steve@iris5.icr.ac.uk
+On leave from: Chemistry Department, The University of Auckland, Auckland, New Zealand
Hydrogen bond
Distance (Å)
N1...N3A6
3.1
N1...O2T20
2.9
N3...O2T7
3.1
N3...O2T19
3.0
N5...N3A18
3.0
N1...W15
2.8
N7...W8
2.9
W8...O4'A5
3.1
W15...O4'A6
3.0
W15...W24
2.8
W24...O4'G10
3.0
W24...O2C9
3.0
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