Báo cáo Y học: Two 1 : 1 binding modes for distamycin in the minor groove of d(GGCCAATTGG) docx

Two 1 : 1 binding modes for distamycin in the minor grooveof dGGCCAATTGG Koen Uytterhoeven1, Jiri Sponer2and Luc Van Meervelt1 1 Biomolecular Architecture, Department of Chemistry, Katho

Two 1 : 1 binding modes for distamycin in the minor groove

of d(GGCCAATTGG)

Koen Uytterhoeven1, Jiri Sponer2and Luc Van Meervelt1

1

Biomolecular Architecture, Department of Chemistry, Katholieke Universiteit Leuven, Belgium;2Institute of Biophysics,

Academy of Sciences of the Czech Republic, and National Center for Biomolecular Research, Brno, Czech Republic

Single-crystal X-ray structure determinations of the complex

between the minor-groove binder distamycin and

d(GGCCAATTGG) reveal two 1 : 1 binding modes which

differ in the orientation of the drug molecule in the minor

groove The two crystals were grown from different

cry-stallization conditions and found to diffract to 2.38 and

1.85 A˚, respectively The structures were refined to

comple-tion using SHELXL-93, resulting in a residual R factor of

20.30% for the 2.38-A˚ resolution structure (including 46

water molecules) and 19.74% for the 1.85-A˚ resolution

structure (including 74 water molecules) In both

orienta-tions, bifurcated hydrogen bonds are formed between the amide nitrogen atoms of the drug and ATbase pairs With a binding site of at least five base pairs, close contacts between the terminal distamycin atoms and guanine amino groups are inevitable The detailed nature of several of these inter-actions was further investigated by ab initio quantum chemical methods

Keywords: distamycin; drug–DNA complex; minor groove binder; quantum chemical calculations; X-ray structure

Distamycin A (Fig 1) is a member of a family of naturally

occuring oligopeptides showing antiviral and antibiotic

properties Like other minor-groove binder drugs,

distamy-cin binds noncovalently in the minor groove of DNA with a

binding preference for stretches of AT-rich sequences [1],

thereby preventing DNA and RNA synthesis by inhibition

of the corresponding polymerase reaction The crystal

structure determination of a 1 : 1 distamycin–d(CGCAAA

TTTGCG) complex (12-dista) at 2.2 A˚ resolution shows

that the drug covers five of the six ATbase pairs [2] The

amide nitrogen atoms of the drug form hydrogen bonds to

N3(A) and/or O2(T) atoms in the minor groove The

complex is further stabilized by van der Waals’ and

electrostatic interactions

The selectivity for AT-rich sequences of minor-groove

binders was first thought to have sterical reasons: the bulky

NH2 group at the floor of the minor groove of

CG-containing regions can prevent binding of these drugs [3]

More recently, factors such as minor-groove width

influen-cing the extent of van der Waals’ interactions [4] and

electrostatic interactions between the positively charged

drug and the more negatively charged minor groove in the case of ATsequences [5] were added

Solution NMR studies have also discovered side-by-side binding of two distamycin molecules in the minor groove of d(CGCAATTGCG) [6] More structural information about this 2 : 1 binding mode was first provided by the crystal structure of d(ICICICIC)–mycin [7] and later by side-by-side complexes of dista-mycin with natural targets d(ICITACIC), d(ICATATIC) and d(GTATATAC) [8,9] Owing to the overlap of about 75%, the two staggered antiparallel distamycin molecules span almost eight base pairs and are kept together by dipole–dipole interactions between stacking pyrrole rings and amide bonds Each drug hydrogen-bonds with the bases of only one DNA strand and stacks with the sugar rings

We have previously reported the structure determin-ation at 1.9-A˚ resolution of the complex of the shor-ter minor-groove binder 4¢,6-diamidino-2-phenylindole (DAPI) with d(GGCCAATTGG) (10-DAPI), revealing

a novel off-centered binding with a hydrogen bond between the drug and a CG base pair [10] In an attempt

to use similar crystal engineering techniques to improve the resolution of 1 : 1 distamycin–DNA complexes (currently 2.2 A˚ for 12-dista and 2.0 A˚ for the dista-mycin–d(CGCGAATTC+GCG) complex where C+¼ 5-methylcytidine (NDB entry code GDLB41), we have cocrystallized distamycin with the decamer d(GGCCAA TTGG) Intensity measurements for two crystals obtained from different crystallization conditions were carried out

to 2.38 and 1.85 A˚ resolution Whereas for one crystal the distamycin orientation and binding site is the same as in 12-dista, the orientation of the drug is inverted in the other crystal Both orientations show interactions between the drug and guanine NH2 groups For the inverted orientation, the DNA–distamycin interaction is also characterized by ab initio methods

Correspondence to L Van Meervelt, Biomolecular Architecture,

Department of Chemistry, Katholieke Universiteit Leuven,

Celestijnenlaan 200F, B-3001 Leuven (Heverlee), Belgium.

Fax: + 32 16 327990, Tel.: + 32 16 327609,

E-mail: Luc.VanMeervelt@chem.kuleuven.ac.be

Abbreviations: 12-dista, crystal structure of the

d(CGCAAATTTGCG) complex (2); 10-DAPI, crystal structure of

the d(GGCCAATTGG)–DAPI complex (10); MPD, 2-methyl-2,

4-pentanediol; DAPI, 4¢,6-diamidino-2-phenylindole; HF,

Hartree-Fock; MP2, Moeller–Plesset perturbational theory.

Note: a web page is available at

http://www.chem.kuleuven.ac.be/research/bma/

(Received 20 December 2001, revised 10 April 2002,

accepted 23 April 2002)

E X P E R I M E N T A L P R O C E D U R E S

Crystallization and data collection

The DNA decamer d(GGCCAATTGG) was purchased

from Oswel DNA service (University of Southampton, UK),

distamycin from Serva Biochemica (Heidelberg, Germany)

Crystals were grown at 16C using the sitting drop method

from two different conditions containing 54.4/33.25 mM

sodium cacodylate buffer (pH 6.0), 35.0/105.0 mMMgCl2,

70 mM NaCl, 8.8% 2-methyl-2,4-pentanediol (MPD),

10.5 mM spermine, 0.25/0.42 mM ssDNA and 0.125/

0.21 mMdistamycin against a 50/35% MPD stock solution

From a bar-shaped crystal of dimensions 0.4· 0.1 ·

0.05 mm from condition 1, intensity data were collected at

100 K on a MAR345 imaging plate detector at beamline

X11 in an EMBL Hamburg (k¼ 0.9116 A˚) over a 105  u

range with increments of 1.5 using cryocooling techniques

with a crystal-to-detector distance of 350 mm

A well-diffracting crystal of dimensions 0.2· 0.1 · 0.05 mm from condition 2 was mounted for data collection

at 100 K using a similar protocol at beamline BW7b in an EMBL Hamburg (150 u range, crystal-to-detector dis-tance 250 mm, k¼ 0.8423 A˚)

Data were processed using the DENZO/scalepack [11] suite of programs Data collection statistics for both crystals are given in Table 1 The final resolution limit of the diffraction pattern was 2.38 A˚ for crystal 1 and 1.85 A˚ for crystal 2

Structure solution and refinement Unit cell parameters and space group indicated isomorph-ism with the d(GGCCAATTGG)–DAPI structure, which was used as a starting model (NDB entry code DD0002, except DAPI and solvent molecules) for further refinement

on F2using SHELXL-93 [12] The nucleotides of strand 1 are labeled G1–G10 in the 5¢ fi 3¢ direction and G11–G20

on strand 2, and the drug is labeled D After determination

of the weighting factor and positional adjustment of parts of the DNA structure, water molecules were added, but not in the minor-groove region At this stage of the refinement, the distamycin molecule was located in the (Fo) Fc) Fourier difference map In subsequent refinement cycles, more water molecules were gradually added During the conjugate-gradient refinement, no torsion angle or hydrogen-bond restraints were applied The 1,2 and 1,3 distances used as dictionary values for distamycin were based on netropsin [13], except for the formamide end which was based on fragments retrieved from the Cambridge Structural Database [14]

Fig 1 Structure and numbering scheme of distamycin Hydrogen

atoms attached to pyrrole and alkyl groups are not shown.

Table 1 Data collection and refinement statistics of the d(GGCCAATTGG)–distamycin complexes NA, Not available.

Data collection statistics

Unit cell (A˚) a ¼ 26.011, b ¼ 40.861, c ¼ 53.164 a ¼ 25.289, b ¼ 36.439, c ¼ 53.047

25.1 (2.42–2.38 A˚) 19.5 (1.92–1.85 A˚)

93.4 (2.42–2.38 A˚) 96.9 (1.92–1.85 A˚)

Reflections with I > 3 r(I) (%) 80.3 (100.0–2.38 A˚) 80.3 (100.0–1.85 A˚)

46.3 (2.42–2.38 A˚) 55.3 (1.92–1.85 A˚) Refinement statistics

For crystal 1, the R value converged to 20.30% after

addition of 46 water molecules (Rfreewas not used to avoid

further reduction of the number of data per parameter at

this resolution) For crystal 2, the first maps already

indicated an inverted orientation (hereafter called

orienta-tion B) of the drug molecule with respect to crystal 1

(orientation A) Therefore refinement for crystal 2 was

monitored using Rfreecalculated for a reference set of 10%

of the reflections Addition of 29 water molecules and

distamycin led to the following R values: R¼ 23.88%,

Rfree¼ 32.31% for orientation A, and R ¼ 22.79%,

Rfree¼ 30.75% for orientation B Final R values for crystal

2 were R¼ 19.74%, Rfree¼ 28.01% for orientation B An

independent refinement with orientation A resulted in

R¼ 20.30%, Rfree¼ 29.84% As a consequence and in

agreement with the electron density maps, orientation B was

retained for crystal 2

Figure 2 shows the final (2Fo) Fc) electron-density maps

in the minor-groove region for both crystals Refinement

statistics are presented in Table 1

The helical parameters in accordance with the Tsukuba Workshop guidelines [15], and torsion angles were calcula-ted with the 3DNAprogram [16]

Quantum chemical calculations

Ab initio quantum chemical calculations were used to investigate intrinsic molecular interactions of a number of close intergroup contacts observed in the crystal Special attention was given to contacts involving the guanine amino groups Appropriate fragments of the drug and several proximal bases have been taken from the crystal structure Intermolecular positions of the interacting species were frozen based on the crystal data using a set of constraints involving three (in some cases more) appropriate non-hydrogen atoms on each monomer The rest of the structure including all the hydrogen positions was relaxed using gradient optimization This procedure has been extensively used in the past to investigate local contacts seen in DNA crystal structures and allows full relaxation of the electronic structure and hydrogen positions while keeping the systems

Fig 2 Final (F o ) F c ) electron-density maps in the minor groove of the crystal structure of the d(GGCCAATTGG)–distamycin complex in which the drug has been omitted from the final refined model (A) Crystal 1 in orientation A; (B) crystal 2 in orientation B The refined distamycin position is superimposed on the difference density for reference, contouring at 1 r (yellow) and 2 r level (green) The figure was prepared with BOBSCRIPT [34] and 3 [35].

studied in the experimental geometry [10,17–20] The

optimizations were carried out within the Hartree–Fock

(HF) approximation with the standard polarized 6–31G*

basis set Although this level of calculations underestimates

the flexibility of amino groups, it nevertheless is sufficient to

reveal when the amino group is activated by molecular

interactions towards a partial sp3hybridization [21–23]

Interaction energy calculations between drug fragments

and nucleobases were carried out assuming the quantum

chemical-optimized geometries with inclusion of electron

correlation effects using the second-order Moeller-Plesset

perturbational theory (MP2) with the 6–31G basis set

augmented by diffuse d-polarization functions to all

sec-ond-row elements [exponents of 0.25, designated as

6–31G*(0.25)] to properly account for the dispersion

attraction [22,23] The calculations were corrected for the

basis set superimposition error using the standard counter-poise procedure The quantum chemical procedure used in this study allows reliable semiquantitative characterization

of the nature and intrinsic
in vacuo strength of the observed intermolecular contacts All quantum chemical calculations were carried out using theGAUSSIAN94 program suite [24]

R E S U L T S A N D D I S C U S S I O N

Both complexes (Fig 3) have a conformation that closely resembles the native decamer structure [25] and its DAPI complex [10]: a central octamer d(CCAATTGG) consisting

of normal Watson–Crick base pairs is at both ends flanked

by two overhanging guanines forming triplets in the crystal packing All three drug–decamer complexes differ from the native 1.15-A˚ resolution structure in the phosphate

confor-Fig 3 Stereoscopic representation of the

distamycin–d(GGCCAATTGG) complexes.

(A) Crystal 1 in orientation A; (B) crystal 2 in

orientation B The DNA is drawn with open

bonds, and the distamycin with solid bonds.

Nitrogen, oxygen and phosphor atoms are

grey, and carbon atoms are white The figure

was prepared using B OBSCRIPT [34].

mation of residue C13, which correlates with the difference

in resolution as described previously [10] As expected for

B-DNA, the sugar puckering modes for the central octamer

duplex are situated in the normal C3¢-exo to O4¢-endo

range, the southern part (C2¢-endo) of the pseudorotation

cycle However, for the overhanging guanines, C3 and C13,

the puckering modes are closer to those for A-DNA, which

is a consequence of triplet formation

DNA–distamycin interactions

Distamycin binds in the minor groove of the central

octamer The expected binding site of at least five base

pairs makes interactions with GC base pairs inevitable

Both structures not only differ in resolution, but also in the

orientation of the distamycin molecule (Fig 4) In the lower

resolution structure of crystal 1, the orientation

(orienta-tion A) is similar to that described for 12-dista [2] The

higher-resolution structure of crystal 2 shows an inverted

orientation of distamycin (orientation B)

Position of distamycin in the lower-resolution structure

(orientation A, Fig 4A)

Distamycin binds to five base pairs covering the sequence

d(AATTG) The positively charged amidinium end group is

orientated toward the A5.T18 base pair with atom N9(D)

lying deep in the minor groove interacting with N3(A5)

(2.70 A˚) and the partial negative O4¢ atoms O4¢(A5)

(3.29 A˚) and O4¢(A6) (2.80 A˚) A close contact with the

amino group of G19 is avoided (N9(D)…N2(G19) 3.86 A˚)

The nearest neighbor of N8(D) is O4¢(3.41 A˚) Bifurcated

hydrogen bonds are formed between the amide nitrogen

atoms simultaneously to both N3(A6) and O2(T18) for

N7(D), O2(T7) and O2(T17) for N5(D), and N3(A15) and

N2(G9) for N1(D) N3(D) only has one close contact to N3(A16) and is 3.59 A˚ away from O2(T8) At the other end

of distamycin, the formamide O1(D) is oriented away from the groove The nearest neighbor of O1(D) is a sugar O4¢(A15) atom at 3.50 A˚

The three pyrrole rings are rotated to each other to follow closely the curvature of the groove Rings A and B make an angle of 22.9, rings B and C 28.4  and ring C makes an angle of 35.9 with the terminal amidinium group The drug–DNA complex is further stabilized by van der Waals’ interactions mainly between carbon atoms

of the three pyrrole rings and the sugar–phosphate backbone No water molecules are in direct contact with the drug molecule

Position of distamycin in the higher-resolution structure (orientation B, Fig 4B)

It was clear from the density maps of the 1.85-A˚ resolution structure that the drug has to be rotated over 180 with respect to orientation A As a consequence, the amidinium group is now orientated toward the G9–C12 base pair and distamycin now interacts with six base pairs

The N9(D) atom of the amidinium group is in close contact with amino group N2 of guanine G9 (3.16 A˚) and hydrogen-bonded with N3(A15) (2.85 A˚) This N9(D) atom

is also in contact with O2(C14) by two intermediate water molecules

The O1(D) atom of the formamide group now points toward the C4–G19 base pair with a very close contact with N2(G19) of only 2.57 A˚ Atom O1(D) is further in contact with O2(C4) (3.39 A˚), O4¢(A5) (2.94 A˚) and two water molecules (2.77 and 2.95 A˚) In the same formamide group, atom N1(D) interacts with N3(A5) (3.12 A˚) and O4¢(A6) (3.03 A˚)

Fig 4 Schematic view of the distamycin interactions in the minor groove of d(GGCCAATTGG)–distamycin structures (A) Crystal 1; (B) crystal 2.

The nitrogen atoms of the amide linkers between the

pyrrole rings form bifurcated hydrogen bonds with the

thymine or adenine bases The central N5(D) atom of

the drug is hydrogen-bonded to the two O2 atoms of the

thymine bases of the central ATsteps Nitrogen atoms

N3(D) and N7(D) bridge N3(adenine) and O2(thymine)

atoms of opposite strands Furthermore, several stabilizing

van der Waals’ contacts between the O4¢ atoms of the DNA

sugars and distamycin atoms are observed

Rings A and B make an angle of 8.6, rings B and C

21.1, and ring C makes an angle of 15.7  with the terminal

amidinium group

Quantum chemical analysis of the base–distamycin

contacts

For both orientations, the crystal structure reveals a number

of close intermolecular contacts including interactions

between the guanine amino groups and the terminal

distamycin atoms Such amino-group interactions are often

assumed to be repulsive steric clashes To clarify the nature

of the selected interactions, we used quantum chemical

calculations carried out at the MP2/6–31*(0.25)//HF/

6–31G* approximation (see Experimental procedures) As

the interactions were most surprising for the 1.85-A˚

resolution structure, the calculations were only performed

for orientation B Further, such calculations are usually

only performed for systems of resolution 2 A˚ and better, as

outcomes of such calculations may be spoiled by inaccurate

positioning of the interacting groups

The contact area of the distamycin amidinium end was

investigated by evaluating the hydrogen positions and

interaction energy terms for the interactions between the

terminal drug segment and the bases of base pair A15–T8

and base G9 (Table 2, Fig 5) The drug binding to A15 is

very strong, with substantial electrostatic and dispersion

components indicating a strong hydrogen bond between

N9(D) and N3(A15) The intrinsic strength of this

interac-tion is halfway between CG Watson–Crick and AT

Watson–Crick base pairs [22,23] Furthermore the drug

also recognizes T8 by a long-range electrostatic interaction

between N7(D) and O2(T8) The long-range nature of this contact can be deduced considering both the geometry and the negligible correlation component to the interaction energy The interaction with G9 is an energetically neutral van der Waals’ contact with an attractive correlation (dispersion) component and thus a repulsive HF compo-nent This means no active recognition The guanine amino group is certainly influenced by this contact It has a substantial pyramidal character: both hydrogen atoms deviate 0.39 and 0.49 A˚ from the best plane through the base atoms The amino-group nonplanarity is modestly enhanced over isolated guanine optimized by the same method (hydrogen deviations 0.10 and 0.41 A˚, respectively) Note also that, because of increased propeller twisting of the

GC base pair, the cytosine base (its O2 atom) not involved

in the calculation helps to lead the guanine amino-group hydrogen atoms away from the drug A search in the Cambridge Structural Database [14] shows that most amino groups in guanine fragments are almost planar Note, however, that in vacuo the amino group is substantially nonplanar [21–23], and this electronic structure feature may reoccur in DNA when intermolecular interactions profit from the nonplanarity [10,17–19,21–23] This has been observed previously for the DAPI–DNA complex [10] and

is seen to a lesser extent here also

The interaction between the neutral formamide part of the drug at the other end and the proximal base pairs A5–T18 and C4–G19 is less favourable than the amidi-nium–DNA interactions because of the neutral character of this end (Table 2, Fig 5) The composition of the interac-tion energy shows that the drug interacts with the ATpair more in a van der Waals’ than in a hydrogen-bonding manner (Hydrogen-bonded systems are always dominated

by a strong HF component, which could be obtained as the difference between the total interaction energy and the correlation component in Table 2.) The contact with the CG base pair is weakly repulsive, but not enough to prevent binding The contact with C4 is indeed not favorable because of the proximity of the two oxygen atoms O1(D) and O2(C4) (3.39 A˚) Despite the very short distance between O1(D) and N2(G19), the energy components show that this contact is not a strong hydrogen bond at all This

is also clear from the amino-group hydrogen positions, pointing away from O1(D) To obtain an upper limit of the possible attraction that can be achieved by this part of the drug and a CG base pair, we alleviated all constraints during the optimization A completely unconstrained gas phase optimization of the formamide part and base pair C4–G19 illustrates that not much of the hydrogen-bonding potential

is used In the most optimal binding, the interaction energy between G19 and the formamide fragment would be )35.1 kJÆmol)1 (Ecor¼)4.2 kJÆmol)1) However, this optimal geometry is not possible because of the interactions

of the rest of the distamycin with the oligonucleotide We also carried out some additional optimizations with differ-ent constraints (not shown) These confirmed that in the refined crystal geometry the drug is rather too tightly packed against the G19 base The interaction can be improved by locally relaxing the drug geometry, but no hydrogen bond can be obtained close to the experimental geometry It should also be noted that the CG–formamide end interaction might be supported by two crystallographic water molecules (O116 and O126) Although several

Table 2 Total interaction energy and correlation component for the

interaction between terminal distamycin fragments and bases in close

contact with distamycin evaluated at the MP2/6–31G*(0.25)//HF/

6–31G* approximation and utilizing constraints taken from the crystal.

The electron correlation component of the interaction energy includes

the dispersion attraction and corrections to electrostatic and

polar-ization terms.

Base

Interaction energy (kJÆmol)1)

Correlation component (kJÆmol)1) Amidinium end

Formamide end

optimization attempts (not shown) did not result in a

converged structure, the intermediate structures suggest that

the water molecules are actively involved

Influence of the binding of distamycin on the DNA

conformation

One of the most important effects of drug binding on the

DNA conformation is the widening of the minor groove as

illustrated in Fig 6 The width of the groove is measured by

taking the shortest H5¢–H4¢ distances between the two

DNA chains In the native structure, the minor-groove

width is symmetric Both distamycin and DAPI have a

similar asymmetric opening effect: the widening is more

pronounced at the 3¢ end of the first strand Where DAPI

opens the groove over a distance of three to four base pairs,

distamycin has an effect on at least five base pairs The

opening of the groove by distamycin (by  4 A˚) is more

pronounced than with DAPI (1.5 A˚) In the 2 : 1

side-by-side complexes, the minor groove expands to  7.7 A˚ in

order to accommodate two distamycin molecules

Distamycin makes more close contacts with the atoms in

the minor groove in orientation A than in orientation B

(Table 3) For both orientations, the number of contacts

with both strands is almost equal, which was less

empha-sized for 12-dista

The complexation of distamycin has no major effect on

interbase parameters (buckle, propellor twist, and opening)

Fig 6 Comparison of the minor-groove width based on H4¢–H5¢ dis-tances for the native decamer (blue) and its complex with distamycin (black for the 2.38 A˚ and red for the 1.85 A˚ resolution structure) and DAPI (green).

Fig 5 Optimized geometry based on HF/6–31G* calculations of the interaction between (A) the amidinium end and bases A15, T8 and G9, and (B) the formamide end and bases C4 and G19 Intermolecular geometry frozen according to the crystal data Drug fragment atoms are yellow.

and cartesian neighboring base parameters (tilt, roll, twist,

shift slide, and rise) However, some small adaptions such as

the increased propeller twist of G14–G9 in orientation B are

observed, and are necessary to optimize the complexation

Also the base stacking patterns are very similar to those

observed for the native decamer

C O N C L U S I O N S

Both current crystal structures describe the interaction of

distamycin in the minor groove of the central CAATTG

sequence in a 1 : 1 binding mode The present tight crystal

packing due to the triplet formation is not compatible with a

2 : 1 drug binding mode, which requires a much broader

minor groove The opposite drug orientations in the minor

groove are despite the pseudo-twofold symmetry of the

palindromic sequence distinguishable because of the different

triplet formation at both ends of the oligonucleotide The

length of the minor-groove binder makes contacts with the

G-NH2group at both ends of the drug inevitable For the

novel orientation B, analyis of the absolute interaction

energies obtained by quantum chemical methods shows that

the presence of both G-NH2 does not destabilize the

distamycin binding to an extent that it prevents

complexa-tion

The amidinium end of the drug does not recognize G9

actively, but this region optimizes its conformation with

respect to the available space The amidinium fragment
sits

on the G base; as a consequence, the G-NH2group becomes

pyramidal and the propeller twist of base pair G9–C14

increases by 5 compared with the native decamer, helping

the pyramidalization Thus the DNA structure adapts to

host the drug molecule, including a modest amino-group

pyramidalization

The contacts of the atoms O1(D) and N1(D) at the other

side of the drug are more complicated The interaction

energy with A5 of )19.6 kJÆmol)1 is close to those of a

water dimer Combined with the N1(D)…N3(A5) distance

of 3.12 A˚, this could possibly lead to the conclusion that a good hydrogen bond is formed However, the composition

of the interaction energy (the dominating dispersion component) and the N1(D)-H…N3(A5) angle (137 ) do not support this view The short contact between atoms O1(D) and N2(G19) (2.57 A˚) again cannot be classified as

a strong hydrogen bond Optimal hydrogen bonding in this region as located by an unconstrained gas phase optimi-zation is not possible here because of the other DNA– distamycin interactions It is helpful to check the electron density again in this region: both O1(D) and C1(D) are not

in reasonable (2Fo– Fc) electron density, whereas the rest

of the drug molecule fits these maps nicely (Fig 2B) Also the temperature factors of these two atoms are much higher than those of the other distamycin atoms (Fig 7) Most probably, this end of the drug has more than one conformation, the average of which is observed This illustrates the use of quantum chemical calculations in further analysis of crystallographic results, a combination

Table 3 Close contacts of atoms in the minor groove of d(GGCCAATTGG) or d(CGCAAATTTGCG) and distamycin atoms Distances less than 3.6 A˚ are considered close contacts.

d(GGCCAATTGG) + distamycin (orientation A)

d(GGCCAATTGG) + distamycin (orientation B)

d(CGCAAATTTGCG) + distamycin (12-dista)

Fig 7 Temperature factors for distamycin in orientation B (crystal 2) Red are high (B  55 A˚ 2

) and white are low (B  20A˚ 2

) temperature factors The figure was prepared using [34].

that is so far unique in the field of biological

macromol-ecules We plan to investigate this binding mode by an

extensive explicit-solvent molecular dynamics simulation in

the near future

Competition experiments demonstrate that distamycin is

capable of replacing netropsin in its 1 : 1 and 2 : 1

complexes with DNA [26] Compared with netropsin, both

distamycin orientations indeed bind better in the minor

groove [27,28]

Different 1 : 1 binding modes and orientations have been

reported for several minor-groove binders such as netropsin

[27,28] and Hoechst 33258 [29–32] In the case of two

orientations fitting the electron density equally well, one can

conclude that both orientations in the minor groove are

energetically very similar, or that the resolution of the

crystal structure determination is not high enough We have

shown that, for DAPI and distamycin, crystal engineering

techniques may overcome the problem of interpreting

electron-density maps In our case in which both

orienta-tions occur in different crystals, one can also conclude that

the two distamycin orientations in the d(GGCCAATTGG)

minor groove are energetically equivalent However, as the

1 : 1 association of distamycin to AT-rich sequences is

extremely fast [33], why is there no disorder in our

structures? Here the charged character of dystamycin may

play an important role The three orthogonal twofold screw

axes present in the space group P212121in general prevent

similar parts of a molecule being in each others

neighbor-hood Adaption of this packing scheme by the drug without

orientational disorder means that the distance between the

positive distamycin ends is always maximal, a situation that

is electrostatically more favourable

A C K N O W L E D G E M E N T S

This work was partly supported by the European Community–Access

to Research Infrastructure Action of the Improving Human Potential

Programme to the EMBL Hamburg Outstation, contract number:

HPRI-CT-1999-00017, and by the Fund for Scientific Research

(Flanders) We thank the staff of the EMBL Hamburg Outstation

for assistance J S was supported by grant LN00A016 (National

Center for Biomolecular Research), MSMTCR All quantum chemical

calculations were carried out at the Supercomputer Center, Brno.

R E F E R E N C E S

1 Zimmer, C & Wahnert, U (1986) Nonintercalating

DNA-bind-ing ligands: specificity of the interaction and their use as tools in

biophysical, biochemical and biological investigations of the

genetic material Prog Biophys Mol Biol 47, 31–112.

2 Coll, M., Frederick, C.A., Wang, A.H.-J & Rich, A (1987) A

bifurcated hydrogen-bonded conformation in the d(AT) base pair

of the DNA dodecamer d(CGCAAATTTGCG) and its complex

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