Netropsin interactions in the minor groove of d(GGCCAATTGG) studied by a combination of resolution enhancement and ab initio calculations Kristof Van Hecke1, Pham Cam Nam2, Minh Tho Nguyen2 and Luc Van Meervelt1 Biomolecular Architecture, Chemistry Department, K.U.Leuven, Heverlee, Belgium Quantum Chemistry, Chemistry Department, K.U.Leuven, Heverlee, Belgium Keywords B-DNA double helix; base triplets; minor groove binder; netropsin; quantum chemical calculations Correspondence L Van Meervelt, Biomolecular Architecture, Chemistry Department, K.U.Leuven, Celestijnenlaan 200F, B-3001 Leuven (Heverlee), Belgium Fax: +32 16 327990 Tel: +32 16 327609 E-mail: Luc.VanMeervelt@chem.kuleuven.be Note A web page is available at http:// www.chem.kuleuven.be/research/bma/ The structure of the complex between the minor groove binder netropsin and d(GGCCAATTGG) was determined via single-crystal X-ray techniques The structure was refined to completion using refmac5.1.24, resulting in a residual R-factor of 20.0% (including 68 water molecules) Using crystal engineering and cryocooling techniques, the resolution could be ˚ enhanced to 1.75 A, resulting in an unambiguous determination of the drug conformation and orientation As previously noticed, bifurcated hydrogen bonds are formed between the amide nitrogen atoms of the drug and the N3 and O2 atoms of A and T base pairs, respectively, clearly cataloging the structure to class I As the bulky NH2 group on guanine was believed to prevent binding of the drug in the minor groove, the detailed nature of several of the amidinium and guanidinium end contacts were further investigated by ab initio quantum chemical methods (Received April 2005, revised 12 May 2005, accepted 16 May 2005) doi:10.1111/j.1742-4658.2005.04773.x The naturally occurring antiviral and antitumor drug netropsin (Nt) (Fig 1) from Streptomyces netropsis has a binding preference for stretches of AT-rich over GC sequences in the minor groove of double helical B-DNA [1,2] Hence it contributes to the study of DNA base–specific interactions This antibiotic exerts its biological activity by interfering with proteins that regulate replication and transcription processes [3–5] Continuous runs of A or T appear to bind Nt more efficiently than alternating ATAT tracts [6,7], resulting in a preference for binding to the DNA in the order AATT > TAAT ¼ TTAA ¼ ATAT > TATA [8] For AATT tracts, binding of the drug can be divided in two categories [9] In class I structures [10–12] the amide groups form bifurcated hydrogen bonds to N3(A) and O2(T) atoms on opposite strands, thereby displacing the spine of hydration and providing an understanding for the molecular origin of its AT specificity in agreement with earlier results obtained from NMR studies [13] In class II structures [14,15] the Nt is shifted half a base pair, leading to an asymmetrical binding with the amide groups lying in the plane of the base pairs Several class I : complexes between the drug and two DNA strands d(CGCX6GCG) have been Abbreviations 10-DAPI, d(GGCCAATTGG)–DAPI complex crystal structure; 10-Dst, d(GGCCAATTGG)–Dst complex crystal structure; 10mer, d(GGCCAATTGG) crystal structure; 10-Nt, d(GGCCAATTGG)–Nt complex crystal structure; DAPI, 4¢,6-diamidino-2-phenylindole; DFT, density functional theory; Dst, distamycin; HF, Hartree–Fock; MP2, Moeller–Plesset perturbational theory; MPD, 2-methyl-2,4-pentanediol; NAE, netropsin amidinium end; NGE, netropsin guanidinium end; Nt, netropsin FEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS 3531 Netropsin–d(GGCCAATTGG) interactions K Van Hecke et al NH2 H2N NH O HN A N CH3 O HN 10 13 11 B N 12 14 CH3 15 HN O 16 17 H2N 18 10 NH2 Fig Structure and numbering scheme of Nt (only hydrogen atoms attached to nitrogen are shown) determined, where X6 ¼ GA2TBr C [10], A3T3 [11], GT2A2C [16] and a decamer d(CGCA2T2GCG) with a flexible amidinium group at one end of the drug [12] Class II complexes were determined for X6 ¼ GATATC [15] resulting in two structures with Nt in different orientations and [e6G]A2T2C and GA2T2C [14] confirming the ability of Nt to occupy the minor groove in two orientations A side-by-side binding with a guanine base was found in d(CGTATATACG) [17] and a novel end-to-end binding of two Nt molecules was determined for d(CCCCCIIIII), d(CBr5CCCCIIIII) and d(CCCBr5CCIIIII) [18] However, a : complex of Nt with d(CCIICICCII) was also observed [19] The selectivity for AT-rich sequences was first believed to be a consequence of base pair sequence: the additional bulky NH2 group of GC base pairs at the floor of the minor groove was believed to prevent binding and sequence-specific hydrogen bonding [1,20] However indirect sequence alterations are held responsible for AT base pair binding For example, the minor groove width influences the extent of van der Waals’ interactions between the drug and the floor and walls of the minor groove [21,22] Electrostatic interactions between the negatively charged minor groove and the positively charged end groups of the drug are also a key factor in complex formation AT sequences have a more negative minor groove, which can explain the sequence selectivity [23] Modified Nt molecules with the amidinium and guanidinium ends removed, 3532 as well as Nt analogues with cationic ends but with no hydrogen-bonding capabilities, exhibit both an appreciable binding to DNA and a preference for AT base pairs [24,25] Crystal engineering techniques can be used to mimic triple helical fragments in the crystal lattice of d(GGCCAATTGG) [26,27] and at the same time to improve the resolution of the obtained diffraction ˚ data We have previously reported the 1.9 A resolution structure determination of the shorter minor groove binder 4¢,6-diamidino-2-phenylindole (DAPI) with d(GGCCAATTGG [28], revealing a novel off-centered binding with a hydrogen bond between the drug and a CG base pair Structure determinations of the same ˚ ˚ decamer with distamycin (Dst) at 2.38 A and 1.85 A, revealed two : binding modes for Dst in the minor groove [29] Here we report the crystal structure of the same decamer d(GGCCAATTGG) with Nt The decamer d(GGCCAATTGG) forms an octamer B-DNA double helix with two overhanging G bases, which are able to form triple helical fragments; hence the same crystal engineering technique could be used to improve the resolution of the : decamer–Nt complex ˚ to 1.75 A The resolution of DNA–Nt crystal structures ˚ is limited to 2.5–2.2 A, except for the crystal structure ˚ of d(CGTATATACG)–Nt [17] diffracting to 1.58 A In contrast with the previously determined decamer– Dst structure [29], no real short contacts are noticed between the end amidinium and guanidinum end groups and guanine NH2 groups The shortest distance ˚ between N2(G9) and N10(Nt) is 3.32 A The detailed nature of several of the amidinium and guanidinium end contacts is further investigated by ab initio quantum chemical methods Results and Discussion Overall DNA structure The d(GGCCAATTGG)–Nt complex (10-Nt) (Fig 2) has a conformation that closely resembles that of the native decamer (10mer) [27], its DAPI complex (10-DAPI) [28] and its distamycin complex (10-Dst) [29] It consists of a central octamer d(CCAATTGG) duplex with normal Watson–Crick base pairs and two overhanging guanine bases at the 5¢ end of both strands, forming triple helical fragments in the packing The sugar–phosphate backbone of the G11-G12 overhang is a continuous extension of the octamer duplex backbone and forms a parallel triplex fragment with a neighboring duplex within the same column, while the G1-G2 overhang swings out to form an antiparallel triplex with a neighboring duplex of another column FEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS K Van Hecke et al Netropsin–d(GGCCAATTGG) interactions triplex formation and the c-angle of G21 [(+)-ac, 98.2°] due to the end-standing O5¢ The nucleosides all have (–)-anticlinal glycosidic torsion angles (in the range of )94.7° to )135.5°), except for C4 which is (–)-synclinal ()80.5°) and for G2 tending to (–)-synclinal ()87.5°) and due to the flipped out base The sugar puckering is C1¢-exo, except for G2, C4, A5, G9 and G10 which have C2¢-endo puckers In fact all puckers are situated in the southern part of the pseudorotational cycle The average temperature factor is 42.9, 37.2 and ˚ 27.9 A2 for phosphate groups (including O5¢ and O3¢), sugars (excluding O5¢ and O3¢) and bases, respectively The overall temperature factor for the whole structure ˚ (including water) is 35.7 A2 On average, the temperature factors of the Nt atoms ˚ are comparable (ranging from 33.4 to 40.0 A2) with an ˚ average B-value of 35.4 A Position of the Nt molecule in the crystal structure Fig Representation of the d(GGCCAATTGG)–Nt complex, with Nt shown in Corey–Pauling–Koltun The figure was prepared using PYMOL [30] The helical twist is 35.4°, compared to 35.7° for the native 10mer, 35.2° for 10-DAPI and 35.6° for 10-Dst, ˚ ˚ ˚ ˚ and a helical rise of 3.29 A (3.27 A, 3.32 A and 3.25 A for the 10mer, 10-DAPI and 10-Dst, respectively), which fall into the ranges for B-DNA Applying a least squares fit between the 10-Nt struc˚ ture and the native 1.15 A 10mer, 10-DAPI and 10-Dst structures, an overall RMS deviation of 0.97, ˚ 0.59, 0.60 A is obtained, respectively All three DNA–drug complexes differ from the native 10mer in the phosphate conformation of C23 (C13 in the native and DAPI complex), which is correlated with the difference in resolution as explained previously [28] The torsion angles a, b, c and d are in their usual (–)-gauche, trans, (+)-gauche and (+)-anticlinal ranges, respectively Exceptions are found for the b-angle of A5 and C23, which are (+)-ac (144.1° and 140.5°, respectively), the c-angle of G1 and C3 (trans; )171.3° and )173.3°, respectively), which is a consequence of FEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS H–bonds, electrostatic forces and also van der Waals’ interactions play an important role in stabilizing the DNA–drug complex The NH amide atoms of the drug in particular form H-bonds with thymine O2 and adenine N3 atoms in the AATT-rich sequence of the decamer Part of the spine of hydration is substituted by the amide N atoms of the minor groove binder As the crystal engineering technique used resulted in ˚ a 1.75 A resolution, the orientation of the Nt was already undoubtedly clear from the first Fo–Fc difference maps The Nt molecule is positioned squarely in the central AATT region of the minor groove, interact˚ ing (contacts less than 3.3 A) with five bases on strand and with four bases on strand (Fig 3) The Nt molecule sits squarely in the center of the groove, with the pyrrole rings A and B making a dihedral angle (normal perpendicular to planes through the ring atoms) of 40.1° In the crystal structure of the free drug molecule (CSD entry NETRSN) [31] this is only 19.2°, confirming the flexibility around the C6-C9 and N6-C10 bonds to adapt the position of the pyrrole rings to fit approximately parallel to the walls of the minor groove in their own region The terminal guanidinium group is nearly coplanar with pyrrole ring A, as they make a dihedral angle of 6.7° (9.7° for NETRSN) The terminal amidinium group makes a dihedral angle of 12.3° with pyrrole ring B (77.9° for NETRSN, which is a consequence of bending of the amidinium group towards a sulfate ion in the crystal packing) Interestingly, when an idealized Nt molecule is considered (contructed with the gaussview program [32]), 3533 Netropsin–d(GGCCAATTGG) interactions K Van Hecke et al Fig Schematic view of the Nt position and hydration in the minor groove of the d(GGCCAATTGG)–Nt structure Direct drug-base pair hydrogen bonds are shown in red Waters in direct contact with Nt are shown in blue, while waters with non-direct contact are shown in green Only waters with at least two possible hydrogen bonds ˚ less than 3.3 A are shown and further optimized in the gas phase at the density functional theory (DFT) [B3LYP ⁄ 6–31G(d,p)], the optimal dihedral angle between the two pyrrole rings is 22.8° (Table 1) As in the structure of Berman et al [31], the amidinium end was left uncharged, leading to a similar bending of the amidinium group as in the 3534 latter structure (dihedral angle with pyrrole ring B of 63.6°) However, when protonating the amidinium end, an intramolecular hydrogen bond is formed between O3 and H(N9) The guanidinium group is coplanar with pyrrole ring A, with a dihedral angle of 9.3° In the reported structure, the Nt molecule is certainly FEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS K Van Hecke et al Netropsin–d(GGCCAATTGG) interactions Table Dihedral angles (°) between the two pyrrole rings A and B, between the guanidinium end and pyrrole ring A, and between the amidinium end and pyrrole ring B for the 10-Nt reported structure, NETRSN [31] structure and the DFT approximation (B3LYP functional at 6–31G(d,p) basis set), respectively 10-Nt A-B Guanidinium-A Amidinium-B NETRSN DFT 40.1 6.7 12.3 19.2 9.7 77.9 22.8 9.3 63.6 positively charged at both ends, but no intramolecular hydrogen bond occurs at the amidinium end when bonded into the DNA minor groove The calculations show that Nt is flexible at the amidinium end The nonplanarity is reduced in 10-Nt, but remarkably not in the structure of the decamer d(CGCAATTGCG) with Nt (NDB entry GDJ046) [12], which shows a flexible amidinium group similar to the DFT approximation DNA–netropsin interactions and hydration Bifurcated hydrogen bonds are formed between the amide groups and thymine O2 or adenine N3 atoms, clearly cataloging the drug to class I [9] The central ˚ N6(Nt) atom is hydrogen bonded to O2(T27) (3.30 A) ˚ ) Nitrogen atom N4(Nt) bridges and O2(T7) (3.18 A ˚ ˚ O2(T28) (2.77 A) and N3(A6) (3.29 A) N8(Nt) contacts ˚ ) and makes a large contact with O2(T8) (2.84 A ˚ N3(A26) (3.60 A) Also the Nt terminal guanidinium N1 and amidinium N10 atoms are hydrogen bonded ˚ ˚ to N3(A5) (3.21 A) and N3(A25) (3.00 A), respectively Apart from these hydrogen bonds, several stabilizing van der Waals’ interactions between O4¢ atoms of DNA sugars and Nt atoms are observed especially at the amidinium end (Fig 3): N9(Nt) and N10(Nt) both ˚ contact O4¢(A26) (3.30 and 3.29 A, respectively), and ˚ N8(Nt) contacts O4¢(G9) (3.17 A) In the past the bulky NH2 group on guanine was held responsible for preventing binding of the drug In previously reported structures of 10-DAPI and 10-Dst [28,29] close contacts appeared between the amino group, amidinium or formamide nitrogen atoms and N2 guanine atoms However, in the reported structure the closest observed contact distance between an amidinium or guanidinium atom and a N2 guanine atom ˚ (N10(Nt)ỈỈỈN2(G9)) is 3.32 A In 10-DAPI and 10-Dst the contact distance between amino group and amidi˚ ˚ nium atoms and N2(G9) is 3.14 A and 3.17 A, respectively The netropsin makes close contacts with the atoms in the minor groove Twenty one contacts less than FEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS ˚ 3.6 A are observed for strand as well as for strand However, T7 is much more important for contacting Nt than T27 on the opposite strand, while G29 plays a more important role in contacting Nt than G9 on the opposite strand Several water molecules contact Nt (Fig 3) Waters W17 and W54 in particular stabilize the guanidinium and amidinium ends, respectively Nt–solvent–backbone interactions are made by W56, W43 and W40 At the amidinium end, a network of water molecules is observed, extended with contacts to symmetry equivalent water molecules (not shown) Influence on the minor groove width As reported previously [28,29], one of the major consequences of binding of a drug molecule in the minor groove is the widening of this groove, which is confirmed by the reported structure The minor groove width is defined by the shortest H4¢-H5¢ distances between the two opposite DNA strands [22] The minor groove of the native 10mer is symmetrical However, DAPI and distamycin as well as the reported structure all show an asymmetrical widening of this groove, i.e the widening effect is more pronounced at the 3¢ end of the first strand Dst and Nt open the minor groove over a distance of approximately five bases, whereas DAPI opens the groove over a distance of approximately three to four bases The opening of the minor groove is less pro˚ nounced in 10-DAPI (1.8 A) and more pronounced ˚ in the 10-Dst structure (3.4 A) compared to 10-Nt ˚ ) Hence the effect of minor groove opening (2.6 A increases with the size of the minor groove binder The ˚ ˚ average H4¢-H5¢ distance is 5.1 A (10mer), 5.8 A ˚ (10-Nt) and 6.3 A (10-Dst) ˚ (DAPI), 6.1 A Furthermore, the complexation of Nt has no major effect on interbase parameters (buckle, propeller twist and opening) and cartesian neighboring base parameters (tilt, roll, twist, shift, slide and rise) Comparison with other netropsin structures To date, 14 different structures containing Nt have been found in the NDB of which nine contain A and T bases in the central part of the DNA (seven contain an AATT tract and two contain a mixed ATAT tract) Only three structures, GDLB31 [9], GDL014 [11] and GDJ046 [12], are class I structures containing a CAATTG tract and are suitable for comparison with the reported structure Concerning the contacts formed between N and O atoms of the Nt molecule in the different structures and base atoms of the DNA (no 3535 Netropsin–d(GGCCAATTGG) interactions K Van Hecke et al contacts with water molecules are taken into account), the binding of Nt in the reported structure shows most similarity with GDLB31 and GDJ046 Contacts conserved in all four structures are between the N4(Nt) and O2(T28), between the N6(Nt) and O2(T7) and between the N8(Nt) and O2(T8) atoms The guanidinium end most resembles GDJ046 [12] and the amidinium end GDLB31 [9] Probably the similarity with the GDJ046 structure [12] could have been much higher if the amidinium end was not bent towards the DNA in that structure Quantum chemical calculations The netropsin amidinium end The contact area of the Nt amidinium end (NAE) was investigated by evaluating the interaction energies and hydrogen positions of the end fragment and the bases of base pair T8-A25 and base G9 (Table 2, Fig 4) For the bases of the A25-T8 base pair, the interaction energies have a negative sign, hence the complex is more stable than the components in both Hartree– Fock (HF) and correlation parts, suggesting an attractive interaction of Nt with base pair A25-T8 The interaction of the NAE with A25 is very strong with substantial HF and correlation parts, indicating a strong hydrogen bond between N3(A25) ˚ and H(N10) (the N3ỈỈỈH distance is 1.974 A for the optimized structure) However, binding of the drug to A25 is stronger than that to T8 by about 32 kJỈ mol)1 Together with the small correlation component of the interaction with T8 ()26 kJỈmol)1 and )7 kJỈ mol)1 for A25 and T8, respectively) and based on the hydrogen bond geometry [N8(Nt)-HỈỈỈO2 is 169.4°], T8 recognizes Nt by a long-range electrostatic interaction rather than a hydrogen bond (the O2ỈỈỈH distance ˚ is 1.979 A) Table Total interaction energy and correlation component for the interaction between terminal Nt fragments and bases in close contact with Nt calculated at the MP2 ⁄ 6–31G*(0.25)//HF ⁄ 6–31G* level of theory applying constraints according to the crystal geometry Base Total interaction energy (kJỈmol)1) Amidinium end A25 )57 T8 )25 G9 Guanidinium end A5 )72 T28 )55 A6 )16 3536 Correlation component (kJỈmol)1) )26 )7 )33 )33 )23 )13 Fig Optimized geometry based on HF ⁄ 6–31G* calculations of the interaction between (A) the amidinium end and bases A25, T8 and G9; and (B) the guanidinium end and bases A5, T28 and A6 Intermolecular geometry constraint according to the crystal structure The Nt drug fragment is shown in green, nitrogen atoms in blue and oxygen atoms in red The figure was prepared using PYMOL [30] The interaction with G9 shows an attractive correlation component of about )33 kJỈmol)1, but with a repulsive HF component of about 40 kJỈmol)1 The total interaction energy is kJỈmol)1, but in view of the negative value of the correlation component it should be stated that the interaction with G9 is an almost energetically neutral van der Waals’ contact However, it is interesting that the guanine amino group may be influenced by this contact We compared the interaction energies in both a planar and a nonplanar guanine G9 amino group The HF interaction component in the planar guanine amino group was computed to be 55 kJỈmol)1 (40 kJỈmol)1 in the nonplanar guanine amino group), 25 kJỈmol)1 in total interaction energy (7 kJỈmol)1 in the nonplanar guanine amino group) and )30 kJ mol)1 of correlation energy ()33 kJ mol)1 in the nonplanar guanine amino group) A positive sign means a repulsive interaction; hence the tendency of this interaction is to reduce the repulsion by arranging a nonplanar pyramidal NH2 group (Fig 4) When calculating the interaction energy and optimized positions for the hydrogen atoms of water molecule W57, it clearly stabilizes the Nt interaction by hydrogen bonds (data not shown) As noticed previously [29], due to an increased propeller twist of the G9-C24 base pair, the O2(C24) atom (not involved in the calculations) helps to lead the guanine amino group hydrogen atoms away from the drug However, as the propeller twist is only slightly increased ()7.3°, )8.4° and )12.0° for 10mer, 10-Nt and 10-Dst, respectively) this effect is less pronounced for the reported structure FEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS K Van Hecke et al The netropsin guanidium end A strong interaction is observed between the netropsin guanidinium end (NGE) and A5, reflected in both attractive interactions of HF and correlation components This is also a favorable interaction in the case of T28 The composition of the interaction energies shows that the drug interacts with the A5-T28 base pair in a strong hydrogen bond manner [distance (N3)A5ỈỈỈH(N1) ˚ ˚ is 2.006 A and distance O2(T28)ỈỈỈH(N4) is 2.003 A] Also in this case, a water molecule (W17) has a stabilizing effect on the NGE (data not shown) With A6 only a slightly attractive interaction is noticed, with a very small HF component ()3 kJỈmol)1), indicating that this is not a hydrogen bond at all Interestingly, for the NAE contact with the A25-T8 base pair the HF and correlation interactions are more reduced than in the NGE contact with the A5-T28 base pair This is probably due to a self interaction tendency of the amidinium end to form an intramolecular hydrogen bond when it is not constrained according to the crystal geometry The difference in energy in both constrained and nonconstrained cases is about 28 kJỈmol)1 (HF ⁄ 6–31G*) Thus for the NAE in contact with the A25-T8 pair, part of the total interaction energy is used to compensate for the intramolecular hydrogen bond when kept frozen according to the crystal geometry Conclusions The reported crystal structure describes the interaction of Nt in the minor groove of the central CAATTG sequence in a : binding mode The tight crystal packing of d(GGCCAATTGG) due to the triplet formation is obviously not compatible with a : binding mode, which requires a much broader minor groove As for DAPI [28] and Dst [29], we have already shown that resolution enhancement by crystal engineering can overcome the problem of interpreting electron-density maps Indeed, due to the resulting ˚ 1.75 A resolution, the position of Nt could undoubtedly be discriminated from the Fo–Fc difference Fourier maps Bifurcated hydrogen bonds are formed between the amide nitrogen atoms of the drug and the N3 and O2 atoms of A and T base pairs, respectively, clearly cataloging the structure to class I As the additional bulky NH2 group of GC base pairs at the floor of the minor groove was believed to prevent binding and sequence-specific hydrogen bonding, the detailed nature of several of the amidinium and guanidinium end contacts were investigated furFEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS Netropsin–d(GGCCAATTGG) interactions ther by ab initio quantum chemical methods It is clear that Nt can fit into the minor groove of the CAATTG tract, making contact with the bulky amino G9, but without destabilizing the binding to an extent that it prevents complexation The N2(G9)-N1(Nt) contact ˚ (3.32 A) can be considered as an energetically neutral van der Waals’ contact Although the contact is larger ˚ in comparison with 10-Dst (3.17 A) [29], it is still sufficient to influence the amino G9 hydrogen atoms to become pyramidal In fact, the DNA structure adapts to host the drug, by providing a modest G9 amino group pyramidilization The increased propeller twist helps to lead the amino hydrogen atoms of G9 away from the drug However, this effect is less pronounced for Nt than for Dst [29] The minor groove width as defined by the shortest H4¢-H5¢ distances between the two opposite DNA strands [22] shows an asymmetrical widening, i.e the widening effect is more pronounced at the 3¢ end of the first strand This effect is less pronounced in ˚ 10-DAPI (1.8 A) and more pronounced in the 10-Dst ˚ ˚ structure (3.4 A) compared to 10-Nt (2.6 A) Hence the effect of minor groove opening increases with the size of the minor groove binder When fitting the AATT base pair nucleotides of 10-Dst and 10-Nt to each other, it is clear that Dst as well as the Nt drug both ‘sit’ on the G9 amino group Although Dst is a much longer molecule than Nt (number of nonhydrogen atoms), both amidinium ends are almost perfectly aligned It appears that G9 represents some kind of barrier or the end of a binding pocket in the minor groove As a consequence of this barrier and its length, the Dst molecule has to make a much broader turn in the DNA minor groove Experimental procedures Crystallization and data collection The DNA decamer d(GGCCAATTGG) was purchased from Oswel DNA service (University of Southampton, UK) and Nt from Sigma-Aldrich (Bornem, Belgium) Both DNA and Nt were cocrystallized at 16 °C by the sitting-drop vapor-diffusion method from conditions containing 12 mm sodium cacodylate buffer (pH 6.0), 50 mm MgCl2, 0.8 mm spermine, 3.5% 2-methyl-2,4-pentanediol (MPD), 0.2 mm ssDNA and 0.9 mm Nt against a 35% MPD stock solution Crystals, suitable for X-ray diffraction grew in approximately weeks A bar-shaped single-crystal of 0.3 · 0.2 · 0.05 mm3 was used to collect a 98.5% complete data set at EMBL beamline BW7b of the DESY synchrotron in Hamburg Data were collected on a MAR345 imaging plate detector 3537 Netropsin–d(GGCCAATTGG) interactions Table Data collection and d(GGCCAATTGG)–Nt complex refinement Data collection statistics Space group ˚ Unit cell (A) ˚ Resolution (A) Measured reflections Unique reflections Completeness (%) ˚ 20.0–1.75 A ˚ 1.81–1.75 A Rsymm (%) ˚ 20.0–1.75 A ˚ 1.81–1.75 A Multiplicity Mean I ⁄ r(I) v2 Reflections with I > r(I) (%) ˚ 20.0–1.75 A ˚ 1.81–1.75 A Refinement statistics ˚ Resolution range (A) R-value No of nonsolvent atoms No of water molecules ˚ Average B-values of DNA (A2) ˚ Average B-values of Nt (A2) ˚ Average B-values of water molecules (A2) ˚ Rmsd of bond lengths (A) Rmsd of bond angles (°) K Van Hecke et al statistics for the P212121 a ¼ 26.025, b ¼ 38.559, c ¼ 53.203 1.75 61132 5724 98.1 96.2 4.0 19.9 10.7 20.1 1.027 80.7 55.6 9.6–1.75 19.97 441 68 34.0 35.4 46.2 0.021 2.98 (Marresearch GmbH, Norderstedt, Germany) with k ¼ ˚ 0.8457 A, / range ¼ 152°, increment ¼ 2° and crystal-todetector distance ¼ 273 mm at 100 K using cryocooling techniques A total of 5724 unique reflections were observed ˚ in the resolution range of 20–1.75 A (80.7% have I above r(I)) with Rsym ¼ 0.040 Data were processed using the DENZO ⁄ Scalepack suite of programs [33] Data collection statistics are given in Table Structure solution and refinement As unit cell parameters and space group indicated isomorphism with the d(GGCCAATTGG-DAPI) structure [28], this structure (NDB entry code DD0002, with DAPI and all solvent molecules omitted) was used as a starting model for further refinement on F using refmac5.1.24 [34] program from the ccp4 suite [35] with a maximum likelihood refinement target In the initial stage of refinement the R-value was already 33.82% Prior to Nt addition, water molecules were added but not in the minor groove region, by use of the arp ⁄ warp program [36] from the CCP4 suite After a next cycle 3538 Fig Final (2Fo-Fc) electron-density maps contoured in the minor groove of the crystal structure of the d(GGCCAATTGG)–Nt complex at r (cyan) and r (blue) level The refined Nt position is superimposed on the density for reference The figure was prepared using PYMOL [30] Nitrogen atoms are shown in blue, oxygen atoms in red of refinement and addition of 11 water molecules, the Rvalue decreased to 26.69% At this stage of the refinement, it was certain that the Nt molecule was located in the minor groove and the orientation could be undoubtedly discriminated from the (Fo–Fc) Fourier difference map After fitting the Nt molecule in the minor groove region and a further cycle of refinement, the R-value dropped to 24.20% In subsequent refinement cycles, more water molecules were gradually added A total of 68 water molecules were detected in the asymmetric unit, leading to a final R-value of 19.97% Neither mono- or bivalent ions nor spermine molecules could be identified Refinement statistics are present in Table and Fig shows the final (2Fo–Fc) electron-density map in the minor groove region The nucleotides of strand are labeled G1–G10 in the 5¢ to 3¢ direction and G21-G30 on strand The netropsin molecule is labeled NT The atomic coordinates and structure factors have been deposited in the Protein Data Bank FEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS K Van Hecke et al (entry code 1Z8V) Helical parameters in accordance with the Tsukuba Workshop guidelines [37] and torsion angles were calculated with the program 3dna [38] Netropsin–d(GGCCAATTGG) interactions of Scientific Research (Flanders) We thank the staff of the EMBL Hamburg Outstation for assistance P.C.N was supported by the K.U Leuven Research Council (GOA, Doctoral Scholarships) Quantum chemical calculations Optimizations of an isolated Nt molecule (constructed using gaussview [32]) in gas phase were performed at the DFT with the B3LYP functional at 6–31G(d,p) Interactions of the observed contacts between amidinium and guanidinium end fragments of Nt and proximal bases were investigated using ab initio quantum chemical calculations Intermolecular positions of the appropriate fragments were kept frozen based on the crystal structure by constraining appropriate nonhydrogen atoms (as few as possible to maintain the crystal geometry) on each monomer The rest of the structure including all hydrogens was optimized within the HF approximation with the standard polarized 6–31G* basis set This procedure has been used extensively 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 studied in the experimental geometry [28,40– 42,29,39] Although this level of calculation underestimates the flexibility of amino groups it is nevertheless sufficient to reveal amino groups being activated towards an sp3 hybridization [43–45] The interaction energies of the Nt end fragments and proximal bases have been evaluated using the supermolecular method assuming the optimized geometries The interaction energy is therefore determined as the difference between the energy of the complex and energies of the isolated subsystems forming the complex [44] Electron correlation effects are included using the second-order Moeller–Plesset perturbational theory (MP2) with the 6–31G basis set augmented by diffuse d-polarization functions to all second-row elements [exponents of 0.25, designated as 6–31G*(0.25)] to properly account for the dispersion attraction [28,29,44,45] The contact area of the NAE has been studied by evaluating the effect of adenine A25, thymine T8, guanine G9 and one water molecule, W57, on the change of the total energy The NGE, has also been examined whose interaction energies with adenine A5, thymine T28, adenine A6 and water molecule, W17, were determined All quantum chemical calculations were carried out using gaussian 03 [32] Acknowledgements This work was supported by the European Community Research Infrastructure Action under the FP6 ‘Structuring the European Research Area Programme’ contract number RII3 ⁄ CT ⁄ 2004 ⁄ 5060008 and by the Fund FEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS References Wartell RM, Larson JE & Wells RD (1974) Netropsin: a specific probe for A-T regions of duplex deoxyribonucleic acid J Biol Chem 249, 6719–6731 Zimmer C & Wahnert U (1986) Nonintercalating DNAbinding 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Sponer JE & Spackova N (2000) Local conformational variations observed in B-DNA crystals not improve base stacking Computational analysis of base stacking in d(CATGGGCCCATG)2 B M A intermediate crystal structure Nucleic Acids Res 24, 4893–4902 43 Sponer J & Hobza P (1994) Nonplanar geometries of DNA bases Ab initio second order Moller-Plesset study ă J Phys Chem 98, 31613164 FEBS Journal 272 (2005) 3531–3541 ª 2005 FEBS Netropsin–d(GGCCAATTGG) interactions 44 Hobza P & Sponer J (1999) Structure, energetics, and dynamics of the nucleic acid base pairs: nonempirical ab initio calculations Chem Rev 99, 3247–3276 45 Sponer J, Leszczynski J & Hobza P (2001) Electronic properties, hydrogen bonding, stacking and cationbinding of DNA and RNA bases Biopolymers 61, 3–21 3541 ... contact area of the Nt amidinium end (NAE) was investigated by evaluating the interaction energies and hydrogen positions of the end fragment and the bases of base pair T8 -A2 5 and base G9 (Table... base pair binding For example, the minor groove width in? ??uences the extent of van der Waals’ interactions between the drug and the floor and walls of the minor groove [21,22] Electrostatic interactions. .. calculations of the interaction between (A) the amidinium end and bases A2 5, T8 and G9; and (B) the guanidinium end and bases A5 , T28 and A6 Intermolecular geometry constraint according to the crystal structure