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 furth
Trang 1of d(GGCCAATTGG) studied by a combination of
resolution enhancement and ab initio calculations
Kristof Van Hecke1, Pham Cam Nam2, Minh Tho Nguyen2and Luc Van Meervelt1
1 Biomolecular Architecture, Chemistry Department, K.U.Leuven, Heverlee, Belgium
2 Quantum Chemistry, Chemistry Department, K.U.Leuven, Heverlee, Belgium
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 do alternating ATAT tracts [6,7],
resulting in a preference for binding to the DNA
in the order AATT > TAAT¼ TTAA ¼ ATAT >
TATA [8]
For AATT tracts, binding of the drug can be divi-ded 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 spe-cificity 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 1 : 1 complexes between the drug and two DNA strands d(CGCX6GCG) have been
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/
(Received 7 April 2005, revised 12 May
2005, accepted 16 May 2005)
doi:10.1111/j.1742-4658.2005.04773.x
The structure of the complex between the minor groove binder netropsin and d(GGCCAATTGG) was determined via single-crystal X-ray tech-niques The structure was refined to completion using refmac5.1.24, result-ing in a residual R-factor of 20.0% (includresult-ing 68 water molecules) Usresult-ing 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 inves-tigated by ab initio quantum chemical methods
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.
Trang 2determined, where X6¼ GA2TBr2 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 X6¼
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]
How-ever, a 1 : 1 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
respon-sible 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,
as well as Nt analogues with cationic ends but with no hydrogen-bonding capabilities, exhibit both an appreci-able 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 1 : 1 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 1 : 1 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 NH2groups 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 over-hang swings out to form an antiparallel triplex with a neighboring duplex of another column
N7
13 10 11
15
HN
6 9 6
O2
12
HN8
O3
16
17 18
NH 2
10
H 2 N
9
N
5 7 4
5
HN4
3 2
O1
NH
3 1
NH22
H 2 N
1
CH3
14
CH 3
8
A
B
Fig 1 Structure and numbering scheme of Nt (only hydrogen
atoms attached to nitrogen are shown).
Trang 3The 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
ran-ges, 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
triplex formation and the c-angle of G21 [(+)-ac, 98.2] due to the end-standing O5¢
The nucleosides all have (–)-anticlinal glycosidic tor-sion angles (in the range of )94.7 to )135.5), except for C4 which is (–)-synclinal ()80.5) and for G2 tend-ing 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 A˚2 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 A˚2
On average, the temperature factors of the Nt atoms are comparable (ranging from 33.4 to 40.0 A˚2) with an average B-value of 35.4 A˚2
Position of the Nt molecule in the crystal structure
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 differ-ence 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
1 and with four bases on strand 2 (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 guani-dinium 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 con-sidered (contructed with the gaussview program [32]),
Fig 2 Representation of the d(GGCCAATTGG)–Nt complex, with
Nt shown in Corey–Pauling–Koltun The figure was prepared using
PYMOL [30].
Trang 4and 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
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
Fig 3 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.
Trang 5positively 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˚)
and O2(T7) (3.18 A˚) Nitrogen atom N4(Nt) bridges
O2(T28) (2.77 A˚) and N3(A6) (3.29 A˚) N8(Nt) contacts
O2(T8) (2.84 A˚) and makes a large contact with
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
ami-dinium or guaniami-dinium atom and a N2 guanine atom
(N10(Nt)ÆÆÆN2(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˚,
respect-ively
The netropsin makes close contacts with the atoms
in the minor groove Twenty one contacts less than
3.6 A˚ are observed for strand 1 as well as for strand 2 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–back-bone interactions are made by W56, W43 and W40
At the amidinium end, a network of water molecules is observed, extended with contacts to symmetry equival-ent water molecules (not shown)
Influence on the minor groove width
As reported previously [28,29], one of the major conse-quences of binding of a drug molecule in the minor groove is the widening of this groove, which is con-firmed 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 propro-nounced
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 The average H4¢-H5¢ distance is 5.1 A˚ (10mer), 5.8 A˚ (DAPI), 6.1 A˚ (10-Nt) and 6.3 A˚ (10-Dst)
Furthermore, the complexation of Nt has no major effect on interbase parameters (buckle, propeller twist and opening) and cartesian neighboring base parame-ters (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
Table 1 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
struc-ture, NETRSN [31] structure and the DFT approximation (B3LYP
functional at 6–31G(d,p) basis set), respectively.
Trang 6contacts with water molecules are taken into account),
the binding of Nt in the reported structure shows most
similarity with GDLB31 and GDJ046 Contacts
con-served 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
interac-tion energies have a negative sign, hence the complex
is more stable than the components in both Hartree–
Fock (HF) and correlation parts, suggesting an
attract-ive 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 N3ÆÆÆH 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 kJÆ
mol)1 Together with the small correlation component
of the interaction with T8 ()26 kJÆmol)1 and )7 kJÆ
mol)1 for A25 and T8, respectively) and based on the
hydrogen bond geometry [N8(Nt)-HÆÆÆO2 is 169.4],
T8 recognizes Nt by a long-range electrostatic
interac-tion rather than a hydrogen bond (the O2ÆÆÆH distance
is 1.979 A˚)
The interaction with G9 shows an attractive corre-lation component of about )33 kJÆmol)1, but with a repulsive HF component of about 40 kJÆmol)1 The total interaction energy is 7 kJÆmol)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 com-pared the interaction energies in both a planar and a nonplanar guanine G9 amino group The HF interac-tion component in the planar guanine amino group was computed to be 55 kJÆmol)1 (40 kJÆmol)1 in the nonplanar guanine amino group), 25 kJÆmol)1 in total interaction energy (7 kJÆmol)1 in the nonplanar guan-ine 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 opti-mized positions for the hydrogen atoms of water mole-cule W57, it clearly stabilizes the Nt interaction by hydrogen bonds (data not shown)
As noticed previously [29], due to an increased pro-peller 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
Table 2 Total interaction energy and correlation component for the
interaction between terminal Nt fragments and bases in close
con-tact 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
(kJÆmol)1)
Correlation component (kJÆmol)1)
Amidinium end
Guanidinium end
Fig 4 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 struc-ture The Nt drug fragment is shown in green, nitrogen atoms in blue and oxygen atoms in red The figure was prepared using
PYMOL [30].
Trang 7The 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
compo-nents 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)A5ÆÆÆH(N1)
is 2.006 A˚ and distance O2(T28)ÆÆÆH(N4) is 2.003 A˚]
Also in this case, a water molecule (W17) has a
stabi-lizing effect on the NGE (data not shown)
With A6 only a slightly attractive interaction is
noticed, with a very small HF component ()3 kJÆmol)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
intramole-cular 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 kJÆmol)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
intra-molecular 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 1 : 1 binding mode The tight crystal
packing of d(GGCCAATTGG) due to the triplet
for-mation is obviously not compatible with a 2 : 1
bind-ing 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
undoubt-edly 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
cata-loging 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
bond-ing, the detailed nature of several of the amidinium
and guanidinium end contacts were investigated
fur-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 suffi-cient 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)
sitting-drop vapor-diffusion method from conditions con-taining 12 mm sodium cacodylate buffer (pH 6.0), 50 mm
(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 2 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 beam-line BW7b of the DESY synchrotron in Hamburg Data were collected on a MAR345 imaging plate detector
Trang 8(Marresearch GmbH, Norderstedt, Germany) with k¼
0.8457 A˚, / range¼ 152, increment ¼ 2 and
techniques A total of 5724 unique reflections were observed
in the resolution range of 20–1.75 A˚ (80.7% have I above
3 r(I)) with Rsym¼ 0.040 Data were processed using the
DENZO⁄ Scalepack suite of programs [33] Data collection
statistics are given in Table 3
Structure solution and refinement
As unit cell parameters and space group indicated
iso-morphism 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
likeli-hood 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⁄
warpprogram [36] from the CCP4 suite After a next cycle
of refinement and addition of 11 water molecules, the R-value 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 mole-cules were gradually added A total of 68 water molemole-cules 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 3 and Fig 5 shows the final (2Fo–Fc) electron-density map in the minor groove region
The nucleotides of strand 1 are labeled G1–G10 in the 5¢
to 3¢ direction and G21-G30 on strand 2 The netropsin molecule is labeled NT The atomic coordinates and struc-ture factors have been deposited in the Protein Data Bank
Table 3 Data collection and refinement statistics for the
d(GGCCAATTGG)–Nt complex.
Data collection statistics
b ¼ 38.559,
c ¼ 53.203
Completeness (%)
R symm (%)
Reflections with I > 3 r(I) (%)
Refinement statistics
Average B-values of water molecules (A˚2) 46.2
Rmsd of bond angles () 2.98 Fig 5 Final (2Fo-Fc) electron-density maps contoured in the minor
groove of the crystal structure of the d(GGCCAATTGG)–Nt complex
at 1 r (cyan) and 2 r (blue) level The refined Nt position is superim-posed on the density for reference The figure was prepared using
PYMOL [30] Nitrogen atoms are shown in blue, oxygen atoms in red.
Trang 9(entry code 1Z8V) Helical parameters in accordance with
the Tsukuba Workshop guidelines [37] and torsion angles
were calculated with the program 3dna [38]
Quantum chemical calculations
Optimizations of an isolated Nt molecule (constructed using
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
calcula-tions Intermolecular positions of the appropriate fragments
were kept frozen based on the crystal structure by
con-straining appropriate nonhydrogen atoms (as few as
poss-ible to maintain the crystal geometry) on each monomer
The rest of the structure including all hydrogens was
opti-mized 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
hybrid-ization [43–45]
The interaction energies of the Nt end fragments and
proximal bases have been evaluated using the
supermole-cular method assuming the optimized geometries The
inter-action 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
func-tions to all second-row elements [exponents of 0.25,
desig-nated 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
evalu-ating 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
interac-tion energies with adenine A5, thymine T28, adenine A6
and water molecule, W17, were determined
All quantum chemical calculations were carried out using
gaussian03 [32]
Acknowledgements
This work was supported by the European Community
Research Infrastructure Action under the FP6
‘Struc-turing the European Research Area Programme’
con-tract number RII3⁄ CT ⁄ 2004 ⁄ 5060008 and by the Fund
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)
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