DNA-Drug Interactions The Crystal Structure of d(CGATCG) Complexed With Daunomycin

8/7/2019 DNA-Drug Interactions The Crystal Structure of d(CGATCG) Complexed With Daunomycin

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Me

Melo0 0. H

Me

OHFigure 1. Molecular formula of daunomycin. Theaglycon chromophore consists of 4 fused rings, A to D,with the daunosamine sugar attached to ring A.

shown that the intercalating chromophore may beeither parallel (proflavine) or perpendicular(daunomycin) to the long axis of the DNA base-pairs (Neidle et al., 1977; Quigley et aE., 1980). Thestructure of daunomycin complexed to the self-complementary hexadeoxynucleotide d(CGTACG)provided detailed information on the type of base-pairs that compose the intercalation site and on theoverall geometry o f daunomycin intercalation(Quigley et al., 1980; Wang et al., 1987). Thestructure illustrates three principal functional partsof anthracycline antibiotics; the intercalator (ringsB to D), the anchoring function associated with ringA and the amino sugar.Footprinting experiments in conjunction withstructural studies are essential for the dissection ofthe properties of a drug. In this technique, DNA ofdefined sequence is reacted with a nucleic acid-binding drug and the use of various nucleases onthe resulting complex makes it possible to deter-mine preferred binding sites. Results from thefootprinting of triostin A and echinomycin (vanDyke & Dervan, 1984; Low et al., 1984a,b)supporting the X-ray-derived results of complexeswith d(CGTACG) (Wang et aE., 1984; Ughetto et al.,1985) are noteworthy in this respect. Footprinting(Phillips et al., 1978; Chaires et al., 1987) andtheoretical (Pullman, 1987) studies indicate thatsequence specificity of the daunomycin-DNA inter-action cannot be described in terms of the two

base-pa.irs c*omprising the int,erc:alat,ictn sitta. 21quantitative explanation can be obtai~tetl only b>models in which a triplet of base-pairs is regarderlas the recognition sequence of the drug. Foot-printing experiments (Chaires et al.. 1987) haveindicated that the preferred daunomycin tripletbinding site contains adjacent G. C base-pairsflanked by an A VT base-pair. A theoretical study(Pullman, 1987) of model trideoxynucleotides led tothe arrangement, illustrated by Figure 2, of tripletbase-pairs in order of decreasing affinity fordaunomycin. We have determined the crystalstructure of the d(CGATCG)-daunomycin complexto establish any neighbouring sequence effects onthe intercalation of daunomycin.

2. Materials and Methods(a) Synthesis, crystallization and data collection

The complementary oligomer d(CGATCG) was synthe-sized on an Applied Biosystems DNA synthesizer (381A)by phosphoramidite methodology (McBride & Caruthers,1983), and purified by ion-exchange liquid chromato-graphy and reverse-phase high-pressure liquid chromato-graphy. Daunomycin hydrochloride, purchased fromSigma Chemical Co., was used without purification.Crystallization was carred out in Corning depression glassplates by the vapour diffusion method (McPherson, 1982).Crystals suitable fo r diffraction were obtained over aperiod of weeks at 20C from a solution containingapprox 1.5 mw-single-stranded d(CGATCG). 1.5 mM-daunomycin hydrochloride, 20 mM-magneSiUUI acetate,3.5 mw-spermine hydrochloride? 20 mlcl-sodium caco-dylate (pH 6.5), 9% (v/v) 2-methyl-2,4-pentanediol byequilibration with a reservoir of 50% (v/v) 2-methy l-2.4-pentanediol. All diffraction data were obtained from 2red, tetragonal, rod-shaped crystals. Each crystal wassealed in a gla.ss capillary and maintained at 4C in orderto minimize its deterioration. Precession photography ofthe 1st crystal (0.37 mm x 0.50 mm x 0.90 mm) indicateda tetragonal system, t4,2,2 or P4,2,2, which suggestedstructura,l isomorphism with the d(CGTACG)--daunomycin complex studied previously (Quigley et al.,1980; Wang et al., 1987). Cell dimensions of a = 27.98 Aand c = 52.87 A were obtained. and 3-dimensional datawere collected in w scan mode on a Syntex P2,diffractometer equipped with a graphite monochromator(wavelength = 1.5418 A)> a long arm and helium path.The reflections in a complete octant (0 < h, 0 I k. 0 5 I) ofthe reflection sphere out to 2 L%were recorded from the1st rryst.al. A 2nd crystal (O-50 mm X 0.50 mmx 1.00 mm) was used to measure reflections to I.5 A.Merging t.he 2 d&a sets yielded a total of 3529 uniquereflections ( Hmerge0+9), of which 2801 w-ere observed atthe o(F) level. (rystal mosaicitg varied fromAoliZ ~0.4 to 05. whrrr AU,,, is the half-height peakwidth of the w scan. All intensities were corrected fol

INTERCALATION 3 G&r3 5 G-C A-T A-T G-C A-TSITE =a 2 > > 2 >CS-G2 C-G C-G T-A C-G T-AT4-A3 A-T T-A A-T G-C G-C5 3

Figure 2. Triplets of base-pairs arranged in order of decreasing preference for daunomyein intercalation (from atheoretical study by Pullman, 1987).


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DNA-Drug Interactiow 695

Lore&z polarization but crystal deterioration was toosevere for reliable absorption corrections to be made.(b) Structure refinement

The co-ordinates of d(CGTACG)-daunomycin (Wang etal., 1987) were used as a starting model for refinement.Initially, this model was refined as a rigid body with thed(CGATCG)-daunomycin data using a modified versionof SHELX (Sheldrick, 1976). Refinement converged atR = 0.36 fo r 735 reflections in the 8 to 2.5 A range. Atthis stage, electron density (2Fo-FF,) and difference(F,- F,) maps were calculated and displayed on anEvans and Sutherland PS3.50 using FRODO (Jones,1978). On the graphic system, adenine and thymineheterocycle base positions in the helix were exchangedand adjustments of the model co-ordinates were made toimprove its fit with the electron density. Furtherrefinement was carried out by the restrained least-squaresmethod o f Hendrickson & Konnert (1981) using theprogram NUCLSQ (Westhof et al., 1985). In order toprevent. bias of the drug conformation towards thatobserved in the d(CGTACG)-daunomycin complex,refinement was initiated using bond length, chiral volumeand planarity values obtained from the crystal structureof daunomycin in the absence of DNA (Courseille et al.,1979).Restrained refinement of positional and thermalparameters, using the initial drug conformation and withno solvent molecules included, converged at a residual of0.32 with all 2a(F) data. Pu umerous fragment difference,difference and electron density maps were calculated toguide appropriate changes that were required in thedictionary of daunomycin restraints. Alterations in bothnon-planar A ring conformation and orientation of thecyclic amino sugar relative to the aglycone chromophorewere made. After any geometry change had been made,bond length, chiral volume and planarity values wererecalculated and used in the dictionary of restraints forsubsequent. refinement.Difference.and electron density maps were calculatedto find solvent molecules. Acceptance criteria of solventmolecules located from these density maps were similar tothose used previously in this laboratory (Hunter et al.,1986). A total of 40 solvent positions were identified, noneof which could be treated unambiguously as a cation. Thefinal crystallographic residual was 0.25 (weightedresidual = 0.22) for 1845 50(F) reflections in the range 10to I .5 A. A residual of @19 for 678 6a(F) reflections in thelower range, 10 to 2.5 A, indicated that the solventcontribution had been well catered fo r. A residual of 0.40obtained in P4,2,2 confirmed P4,2,2 as the space group.The correctness o f this structure is indicated by anexcellent fit of atomic co-ordinates t.o the eIect.rondensity, an illustration of the aglycone chromophoreelectron density is given in Fig. 3.

3. Results and DiscussionAtomic co-ordinates have been deposited at the

Cambridge Crystallographic Database.(a) Global conformation and crystal packing

The asymmetric unit consists of a single strand ofd(CGATCG) with one molecule of daunomycin. Twosuch units, related by a crystallographic dyad, form

a distorted right-handed B-DNA-like duplex withWatson-Crick base-pairs. A daunomycin moleculeis intercalated at each d(CpG) step (Fig. 4).Kucleotides are labelled Cl to G6 in the Fi to 3direction on strand 1 and C7 to G12 in the 5 to 3direction on symmetry-related strand 2. The twodaunomycin molecules are numbered D13 and D14.The atomic nomenclature used for the daunomycinmolecule is given in Figure 1. Unique solventmolecules are labelled as Wl to W40, andsymmetry-related molecules as W41 to W80,respectively.The overall intercalation geom etry in thed(CGATCG)-daunomycin complex is similar to thatobserved in the d(CGTACG)-daunomycin structure(Quigley et al., 1980; Wang et al., 1987). In thesestructures, head-on insertion of the chromophorebetween d(CpG) base-pairs results in ring Dprotruding into the major groove and semi-saturated ring A, with the amino sugar substituent,lying in the minor groove. DNA-daunomycin directand solvent-mediated hydrogen bonds involvehydroxyl and carbonyl groups at C-9 of theehromophore, the hydroxyl group being essentialfor activity (Henry, 1979; Zunino et al., 1979). Thedaunosamine occupies virtually all t,he minorgroove space, making it difficult to envisageintercalation of more than one drug molecule perthree base-pairs.Inter-duplex interactions between d(CGATCG)-daunomycin molecules are similar to those observedin the d(CGTACG)-daunomycin complex. Thecomplexes stack upon each other along the c-axiswith good overlap of symmetry-related terminalbase-pairs o f adjacent helices. These stackedcolumns are laterally associated by hydrogen bondsbetween O-3(G6(G12)) and 0-lP(T4(TlO)) or(A4(AlO)j.

(b) DNA conformationSugar-phosphate backbone and glycosidic torsionangles in both DNA-daunomycin complexes are

presented in Table 1 together with average valuesfor the dodecamer d(CGCGAATTCGCG) (Dickerson& Drew, 1981). The average torsion angles in bothDNA-drug complexes indicate B-type conforma-tions, similar to that of d(CGCGAATTCGCG), withmodifications in the locality of the drug molecules.These torsion angles illustrate a similar non-symmetrical conformation adopted bv bothdeoxyhexanucleotides on complex formation withasymmetric daunomycin. Although a wider range ofsugar-phosphate torsion ang!es is observed in thed(CGATCG)-daunomycin complex, their averagevalues correspond closely to those found in thed(CGTACG)-daunomycin complex. From the 5 to3 direction, each phosphodiester linkage has a-synclinal (c() -synclinal ([) (IUPAC-IUB, 1983)conformation, except the linkage facing the aminosugar side-chain of daunomycin. The phospho-diester linkages of d(G2pA3). d(G2pT3). andd(C5pG6) have -synclinal (~1) +antiperiplanar (0


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Figure 3. The daunomycin chromophore superimposed on a section of the electron density map (2F, - F,) to illustratethe fit of atomic co-ordinates to the electron densit>

and - clinal (a) antiperiplanar (?J conformations,respectively. Assoc iated with these two con-formations is a change in the /3 angle of A3@= 133; for T3, p = 139) and in the E angle of C5(E = 111; E = - 104 for C5 in the d(CGTACG)-daunomycin complex). The larger differences inindividual torsion angles, especially between thoseof the intercalation sites d(ClpG2) and d(C5pG6),indicate a somewhat more extreme asymmetricconformation adopted by d(CGATCG) than byd(CGTACG) on binding to daunomycin.In d(CGATCG)-daunomycin, glycosyl anglevalues vary from - 71 to - 142. Intercalationbetween bases Cl and G2 results in glycosyl torsionangles of - 142 for Cl and -95 for G2 with an Eangle of - 143 for the d(ClpG2) step, a -anticlinalconformation that is close to the -antiperiplanar

range. In contrast E is - 111 at the d(C5pG6) stepwith glycosyl angles of - 124 and - 71 for C5 andG6, respectively, a - anticlinal conformation closerto the -synclinal conformation. Table 1 illustratesthe similar trend in glycosyl angle values of thed(CGTACG)-daunomycin complex.Intrastrand adjacent phosphorus distances inboth structures are similar at each step, and close tothe typical B-DNA value of 6.7 A.Table 2 presents the amplitude of pucker,pseudorotation phase angle and conventionalnotation for the sugar puckering of each residue.The asymmetry in sugar puckering is similar to thatobserved in the d(CGTACG)-daunomycin complex,suggesting that intercalation of daunomycininfluences the sugar conformations of differentneighbouring base-pairs in a similar fashion.


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DNA-Drug lntera.ction,s

(b)

(clFigure 4. Stereoscopic drawings of the d(CGATCG)-daunomycin complex viewed: (a) perpendicular to the 2-fold axis;(b) into the minor groove along the 2-fold axis; and (c) into t,he major groove along the 2-fold axis. Open and filledbonds are used for the DNA and drug, respectively. Atoms are depicted as spheres of decreasing radii in the orderf>O>N>C.

Base-stacking interactions at each step in both chromophore in d(CpG) is similar in both struc-deoxyhexanucleotide daunomycin complexes areshown in Figure 5, %igure 5(a) illustrates stacking tures, clearly the only d ifference ,is in daunosamineorientation. Base stacking one removed from theinteractions between the chromophore and its intercalation site in both complexes is presented inintercalation site base-pairs. The position of the Figure 5(b). The small translation of the G2 *Cl1


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698 M. II. Moore et al.Table 1Sugar-phosphate backbone and glycosyl torsion angles (deg.)

(CGATCG)- Cl G2 A3 T4 C5 G6 Averagedaunomycin values(CGTACG)- Cl G2 T3 A4 C5 G6daunomycina -71 -57 -66 -104-69 -49 -81 -61B 167 133 -158 142176 139 -178 173Y 60 47 53 29 8946 36 51 59 326 127 130 120 143 122142 146 116 132 143& -143 -133 163 -157 -111-132 -150 -174 -169 -104i -65 164 -67 -89 -169-68 178 -85 -91 172x -142 -95 -132 -196 -124

- 154 -86 -136 -102 -88Average values for d(CGCGAATTCGCG)

-*63 B Y 6171 54 123 -f69Backbone torsion angles are defined as:

p-a-0.5-8-C-5-y-C-4-S-C-3-E-0-3-i-Pand the glycosyl angle as: O-4-C-~-X-N-~-C-~ for pyrimidines andO-4-C-~-Y-N-~-C-~ for purines.Omits the value for A3 or T3.$ Omits the value for C5.

-11 -62-68 -68169 1707174 176t13 4847 45151 132144 137- 1571- 1561-117-81-71 -111-86 -108i-108 --:17

base-pair towards the major groove (observed inboth complexes) contributes to poor purine purineoverlap at the d(G2pA3) step. The most significantdifference in stacking between the two complexes isat the central base-pair step (Fig. 5(c)) and is notrelated to DNA-drug interactions but due tosequence changes. The d(A3pT4) step ind(CGATCG)-daunomycin shows favourable intra-strand stacking in contrast to that observed for thed(T3pA4) step in d(CGTACG)-daunomycin. Thebase-stacking interactions at the d(ApT) andd(TpA) steps in the DNA-daunomycin complexesappear to be very similar to those observed in theabsence of a drug molecule; that is, in the crystalstructure of d(CGCATATATGCG) (Yoon et aZ.,1988). According to the classification proposed byCalladine & Drew (1984), the dinucleotide step ApTis neutral, whereas TpA is a strongly bistable step.These two steps have significantly differentthermodynamic properties, which can be attributed

Table 2Analysis of the sugar conformation

ResiduePuckeringamplitude

Lx)

Pseudorotationphase angle PuckeringW) mode

ClG2A3T4c5G6

33 176 c-2 end048 143 C-2 end040 109 O-4 mdo46 155 C-2 end032 133 C-l end032 182 c-3 ezo

to their different stacking interactions (for a fulldescription, see Travers & Klug, 1987). In thed(CGTACG)-daunomycin complex, it has beenshown that the mobility of the residues oneremoved from the intercalation sites, T3 and A4,are essentially unchanged from that observed inB-DNA (Holbrook et al., 1988).Twist and rise were calculated for both complexes(Dickerson & Drew, 1981; Dickerson, 1983;Calladine & Drew, 1984). Despite a sequencechange, most of the DNA unwinding associatedwith intercalation occurs between residues two andthree in both complexes. In the d(CGATCG)-daunomycin complex at the d(G2pA3) step, thetwist angle is reduced from an average value of 35to 30. In the d(CGTACG)-daunomycin complex,d(G2pT3) is the most unwound step, twist isreduced from 35 to 32, and the d(T3pA4) step isunwound by 2 relative to the d(A3pT4) step of thed(CGATCG)-daunomycin complex. Previous resultssuggest that some sequences of the type NTAN canbe transiently unwound (Drew et al., 1985). Asexpected, the distance between base-pairs of theintercalation site is large (5.2 A), to accommodatethe drug chromophore. The average rise of the othersteps is 3.2 A. For comparison, a 3.4 A rise and 36twist angle is associated with random sequenceB-DNA (Leslie et al., 1980).

(c) Daunomycin conformation and interactionswith d(CGATCO)The conformation of daunomycin in this inter-calation complex resembles that observed in the


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(CGATCG) - dOunOmycln

(CGTACGI- daunomycrn(a)

G2-Cll A3-TIO

GZ-Cll T3-AI0

(b)Fig. 5.


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700 X. II. Moore et al.A3-TIO T4-A9

T3-AI0 A4-T9

(clFigure 5. Stereo views of the base-stacking interactions observed at each step. Hydrogen bonds are represented asthin lines. (a) Daunomycin and its intercalation site base-pairs in both d(CGATCG)-daunomycin and d(CGTACG)-daunomycin complexes. In each drawing, bonds within the drug molecule are filled to clearly distinguish it fromthe surrounding DNA. Both views are perpendicular to the nucleotide base plane with the Cl . G12 base pair closest tothe viewer. In both structures, the D ring methyl group protrudes into the major groove. Note the major difference is inthe flexible saturated daunosamine. (b) A comparison of the d(G2pA3) step in this complex and the d(G2pT3) step inthe d(CGTACG)-daunomycin complex (Wang et al., 1987). In both complexes, a similar asymmetry of the sugar-phosphate backbone is observed. (c ) The d(A3pT4) step in d(CGATCG)-daunomycin and the d(T3pA4) step ind(CGTACG)-daunomycin. Compare the good intrastrand base-pair overlap of (A3pT4) with the poor overlap ofd(T3pA4). In (b) and (c) the view is perpendicular to the best plane through the lower base-pair with the major grooveto the top of each drawing. Covalent bonds of the base-pair closest to the viewer are filled.

d(CGTACG)-daunomycin complex more closely complexes is presented in Figure 6. A view into thethan its conformation when crystallized in the minor groove of the first four base-pairs of theabsence of DNA. The planar aglycon chromophore d(CGATCG)-daunomycin complex is given inconsists of three fused aromatic rings, B, C and D, Figure 7 to illustrate the hydrogen-bondingwith five substituents (Fig. 1). These are two keto systems. Semi-saturated ring A has a conformationoxygen atoms, O-5 and O-12, two phenolic oxygen similar to that observed in the d(CGTACG)-atoms, O-6 and O-l 1, and a methoxy group daunomycin complex. Torsional angles aroundattached to C-4 and orientated away from O-5 into C19-C20 and C7-C20 are -4 and 6, and inthe major groove solvent region. No deviation from d(CGTACG)-daunomycin they are -4.6 and 7.6,planarity greater than 0.04 A was obtained for B, C respectively. All A ring atoms are almost in-planeor D ring atoms, exocyclic ring-bonded atoms (O-4, (no deviation greater than 0.04 A), except C-9,O-5, O-6, C-7, C-10, O-11 and O-12) and the methyl which is displaced by O-6 A on the same side of thegroup attached to O-4 (C21). There are two aglycone plane as the amino sugar. Bonded to C-9 isintramolecular hydrogen bonds, similar to those the hydroxyl oxygen (essential for activity), O-9,observed in the structure of daunomycin alone almost axial to the aglycon plane and within(Courseille et al., 1979), with distances of 2.5 A and hydrogen-bonding distance of N-3 (2.9 A) and N-22,4 A between O-5 and O-6, and between O-11 and (3.3 A) of G2(G8). Although these hydrogen bondsO-12, respectively. A representation of hydrogen are longer than their equivalents in d(CGTACG)-bonds to daunomycin in the two DNA-drug daunomycin (2.6 A and 2.9 A, respectively), the


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l ,-w 4..N-7(612)

. w3IOh(Gl2) o

Me

(a)

Me

G2)

,w3i 1 w4----. N a f /--,,

(Gl2)Oh ,*= \\ IO-6( G 12 ) o.- \N-7(612) W5 I

OH ( \\I -w2 - --- O-l{ A 4 )w13 1

N-3( A 4 )(b)

Figure 6. Two-dimensional diagrams o f the hydrogen-bonding systems to daunomycin observed in (a) thed(CGATCG)-daunomycin complex, note the 4 direct hydrogen bonds between daunomycin and the nucleotide bases.(b) The d(CGTACG)-daunomycin complex, nomenclature is taken from Wang et al. (1987).


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Figure 7. Stereo diagram o f daunomycin intercalated into d(CGATCG) .howing the intermolecular interactions andhydrogen bonds to water molecules. Four base-pairs of the hexamer are drawn with open bonds and the daunomycinwith filled bonds. Hydrogen bonds have thin filled bonds. Some of the drug functional groups involved are labelled(DO-g. DO-13 and DN-3) and the water molecules (W04, WlO, WI 1. W32 and W47).absence of an acceptor water molecule for theproton on guanine N-2 suggests the same type ofhydroxyl group-specific paired hydrogen bonding inwhich N-2 is the donor and N-3 the acceptor. Thecarbonyl substitutent at C9 is indirectly hydrogen-bonded via a bridging water molecule W07 (W47) toO-2 of Cl on the opposite side of the aglycon ring.Hydrogen-bonding distances of 3.0 A and 3.1 A arefound between the carbonyl oxygen and W07 (W47)and between W07 (W 47) and O-2 Cl(C7), respec-tively. O-7 is in an axial position, displaced towardsthe sugar-phosphate backbone so t,hat no intra-molecular hydrogen bond can exist between O-7and O-9 atoms.A least-squares fit of the daunomycin moleculesin the two complexes gives a difference of 0.25 A inthe average atomic positions. The differences in thechromophore region are less than 0.1 8, whereas inthe daunosamine sugar some differences are 1.0 ,%.Major differences are thus in the flexible saturatedregion of the drug. Although it has been suggestedthat the sugar amino group K-3 is involved inelectrostatic interactions with the nucleic acidphosphate group (Gabbay et nl., 1976), in thisstructure N-3 is hydrogen-bonded to O-2 of TlO(T4; 2.8 A) and to a water molecule W50 (WlO;2.9 8). There is also a hydrogen bond between N-3and O-2 of Cl1 (C5; 3.0 A). In d(CGTACG)-daunomycin, the amino sugar N-3 interacts withtwo minor groove water molecules and O-2 ofCll(C5).Another important drug-DNA hydrogen-bondingsystem involves the chromophore substituents O-4and O-5 in the m ajor groove (Fig. 7). A watermolecule (W4) is within hydrogen-bonding distanceof O-4 (D13(1)14); 3.0 A), O-5 (D13(D14): 3.0 A),N-7(G12(G6); 3-O -4) and W3(W 43; 2.3 A). Theequivalent peak in the d(CGTACG)-daunomycinstructure was identified as a sodium ion, because itis octahedrally surrounded by O-4(D14; 3-O A),O-5(D14; 3.1 A), N-7(G12; 2.8 A), W5 (2.8&, W4(3.0 A) and W3 (2*9A) (Fig. 6). The latter twowater molecules are believed to stabilize theposition of the sodium ion by bridging hydrogenbonds to O-lP(G12) and O-6(G12). Althoughcontacts similar to t,he first, four deta iled above were

located in this structure, the two stabilizing watermolecules that com plete t,he octahedral co-ordination did not appear in any density maps. Thecentral peak was therefore treated as a watermolecule (W4).In addition to the hydrogen-bonding systemsdescribed, the complex is stabilized by numerousnon-bonded van der Waals interactions (distances< 3.4 A) between daunomycin and d(CGATCG),particularly in the daunosamine region. Thehydrogen bond between N-3 of the amino sugar ofthe drug and O-2 of thymine has brought moreatoms into close contact with the drug than werefound for the d(CGTACG)-daunomycin complex.

(d) Hydration and thermal parametersAll 40 water molecutes located in the structurerefinement are listed in Table 3 with their R valuesand possible hydrogen-bonded partners: 600/o ofthese water molecules are in the first hydrationshell. Several hydration sites (W9, W21, W35, W37and W39) are not within a reasonable hydrogen-bonding distance of either the DNA or other sites;however, their electron density is well-defined.

Primary hydration sites are on the ph0sphat.ebackbone. The phosphate groups are individuallyhydrated, as is common in R-type oligonucleotidestructures (Saenger et al., 1986). As previouslydiscussed, there are a number of solvent and oation-mediated DNA-drug interactions. The role of waterand cations in this respect is important in DNA-protein interactions, as indicated in the crystalstructure of the trp repressor-operator complex(Otwinowski et al., 1988). The influence ofhydration is one that must be taken into accountfor reliable theoretical studies, and in the design ofnew therapeutic agents.The thermal parameters provide a qualitativeguide to the mobility of the various components inthis structure. The average B values (AZ) can bearranged in the order: bases (10) < aglycone (15)< furanose sugars (16) < phosphate groups (22)< daunosamine (23). The mobility of the aglyconeportion of the drug is comparable to that of the


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DNA-Drug Interactions

Table 3Thermal parameters and possible hydrogen-bondingpartners of solaent moleculesSolvent

Possible hydrogen- Distancebonded partners t-4

Wlw2 3129N3W4

3030

w5 22W6w7 302 1W8 11

WQ 24WI0 17

WllW12w13252234

w14 21w15 25W16 27w17 29

W18 29WlQ 31

w20 31

w21w22 3031W23 31

W24W25 3120W26 22W27 23W28 31W29 34w30 34w31 21W32 27w33 31w34 31w35 24W36 30w37 32W38 29w39 31w40 31

N-2 (G6) 2.50-2P (T4) 2.9w22 3.20-1P (G6) 3.1w4 2.3N-7 (G6) 3.0O-4 (D13) 3.0O-5 (D13) 3.0W3 2.30-1P (C5) 3.0O-5 $5) 3.3W32 2.7O-2 (Cl) 3.1O-13 (Dl4) 3.0N-7 (A3) 3.2w40 3.3w34 3.1N-3 (D13)O-2 (T4)O-4 (D13)N-4 (C5)o-2P (C5)WlQ0-1P (T4)W24w31O-5 (Cl)W270-1P (G2)0-2P (G2)w20w33o-2P (G6)O-5 (C5)w-L9W130-1P (G2)W23w17

2.93.12.93.33.22.93.22.83.22.92.52-Q2.83.32.32.83.13.22.23.23.32.32.3

o-1P (C5) 3.0w2 3-20-2P (A3) 2.5w31 3.1W20 2.3w14 3.20-2P (A3) 3-oO-3 (G2) 3.2o-2P (G6) 3.0W16 2.9O-5 (Cl) 2.8w19 2.2W36 2.6W23 3.1O-5 (D13) 2.6W6 2.7w15 2.9W18 2.8W8 3.1N-7 (G2) 2.9w30 2.6W65O-4 (T4)W8w34

2.83.03.33.1

DNA sugars, whereas the daunosamine is the mostmobile component, even more so than thephosphate groups. This agrees with a local mobilitystudy on the d(CGTACG)-daunomycin complex(Holbrook et al., 1988) referred to earlier.4. Conclusions

It is unclear how daunomycin and its derivativesare able to inhibit DNA replication and transcrip-tion and what role, if any, the sequence specificityof these antibiotics plays, What is known, however,is that this class of antibiotics displays a wide rangeof biological activities and properties not entirelydependent upon the ability to bind DNA. Anyexplanation for the cytotoxic effect of daunomycinmust take into account its interactions withproteins.Consider the following points. There is evidenceto suggest that anthracyclines interfere with thebreakage-reunion reaction of mammalian DNAtopoisomerase II by stabilizing a drug-enzyme-DNA cleavage complex (Tewey et aE., 1984;Pommier et aZ., 1985). Adriamycin (14.hydroxy-daunomycin) is structurally similar todaunomycin and has a similar DNA-bindinggeometry (Wang e t al., 1987). However, this drug isused to treat tumours quite different from thoseagainst which daunomycin is active, and has beenshown to interact with the phospholipid layers ofmembranes (Henry et al., 1985). The search for lesstoxic and more potent derivatives has led to thedevelopment of a wide variety of daunorubicin anddoxorubicin analogues (Acton & Tong, 1981; DuVernay et al., 1979; Israel et al., 1978; Komeshima etal., 1988). In an assay with nicked circular DNAand bacteriophage T4 DNA ligase, no correlationwas obtained between the ability of variousanthracyclines to alter the DNA structure and theircytotoxic or antitumour activities (Montecucco etal., 1988). It was noted, however, that theinhibition of T4 DNA ligase occurs only if theammonium substituent is present at the 3 positionof the sugar. These examples h ighlight the diversebiological studies being carried out on anthracyclineantibiotics which, in conjunction with chemicalstudies, may help us to understand more about thisfamily of drugs. We suggest that a completeunderstanding of the interactions with proteins andthe sequence specificity of these drugs is required inadvance of any rational design of new therapeuticagent)s.The single crystal structure of the d(CGATCG)-daunomycin complex and comparisons with thepreviously reported d(CGTACG)-daunomycin com-plex (Wang et al., 1987) provide information aboutthe specificity of the drugs. In each structure, thegeom etry of daunomycin intercalation is similar.The aglycon chromophore is positioned between thed(CpG) base-pairs oriented at an approximate right-angle to the long dimension of the base-pairs. RingD protrudes into the major groove and the semi-saturated ring A, with its amino sugar substituent.


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704 M. Il. MoorP et, al

lies in the minor groove. Disruption of the DNA duet,o daunomycin binding is localized at the inter-calation site and, in both structures, unwinding isat the adjacent base-pair. The bases of thesenucleotides passively participate in drug binding byproviding functional groups for hydrogen-bondinginteractions. It becomes clear why the d(CpG) siteis preferred fo r intercalation in these complexes. Inthe minor groove, atoms K-3 and N-2 of G2 provideacceptor and donor atoms for hydrogen-bonding tothe hydroxyl O-Q of the drug. The O-2 of Cl isavailable for hydrogen bonding to the carbonylsubstituent of the drug at CQ via a bridging watermolecule. In the major groove, N-7 of G12 and thetwo chromophore substituents O-4 and O-5 aregeometrically positioned to provide the correctenvironment to chelate either a sodium atom or awater molecule. If these features are essential, thenonly the d(TpG) step could provide a similarenvironment for daunomycin intercalation. Such astep has been identified as a possible binding site forthe drug (Chaires et al., 1987). The step d(TpA)might be a less stable intercalation site, due to theloss of the minor groove N-2 when guanine isabsent.The orientation of the drug when complexedprovides a number of features that could disruptDNA-protein interactions and hence interfere withbiological processes. The amino sugar blocks a largesection of the minor groove, is flexible, and hasfunctional groups that could well interact directlywith proteins. In the major groove, a methyl group(C21) is situated in a position that would perhapsmimic a thymine methyl group (Fig. 5(a)).In the d(CGATCG)-daunomycin complex, thecross strand situation of TlO (T4) of the 3neighbouring base-pair relative to G2 results in adirect DNA-drug hydrogen bond between thethymine O-2 and the N-3 of the amino sugar. Sucha hydrogen bond is not present in the complex withd(CGTACG) (Fig. 6). The flexible sugar adjusts tostabilize the d(CGATCG)-daunomycin complex.Given that a direct DNA-drug hydrogen bond mayhave a greater stabilizing effect than a solvent-mediated one then preferred triplet binding sitescould be d(CpGpA), d(TpGpA), d(CpGpG) andd(TpGpG). It is not known what effect, if any, theminor groove amino group would have if a G. Cbase-pair occupies the third position of the bindingsite. A theoretical study indicates that the aminogroup may repulse the daunosamine (Pullman,1987). We suggest that the flexibility of the sugarmay allow a minimization of any such repulsiveforces.Our interest in triplet recognition sites for DNA-daunomycin interactions has been stimulated bythe footprinting results of Chaires et al. (1987)discussed earlier and the study by Brendel et al.(1986), which suggested that a small set of tripletsequences are either over- or underutilized com-pared to their expected random frequency innaturally occurring DNA sequences. It has beenproposed that the sequence specificity of

daunomycin may guide t)he drug int,o thesr specialcontrol regions of the genome, where it. wouldselectively impare replicative events (Chaires et a,/. ,1987). We have initiated a study of these and otherpossible triplet binding sites with a view toexamining any structural differences and, oncedetails are known, hope to explore the use ofpotential energy calculations to improve ourunderstanding of DNA-intercalator interactions.

This study was funded by a Medical Research Councilprogram grant to O.K. We thank Professor A. Wang forco-ordinates, l)r E. Westhof for his computing expertise.Mr Z. Otwinowski. Professor R,. E. Dickerson andtheir colleagues for communicating results in advance ofpublication. We thank Evans and Sutherland for thePS350 graphics system used.

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DNA-Drug Interactions The Crystal Structure of d(CGATCG) Complexed With Daunomycin

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