volume 15 Number 16 1987
Nucleic Acids Research
Molecular modelling of the interactions of tetra-(4-N-methylpyridyI) porphin with TA and CG
sites on DNA
Kevin G.Ford, Laurence H.Pearl and Stephen Neidle*
CRC Biomolecular Structure Unit, Institute of Cancer Research, Sutton, Surrey SM2 5PX, UK
Received June 25, 1987; Revised and Accepted July 27, 1987
ABSTRACT
The molecular structure of the DNA-intercalating ligand tetra-(4-Nmethylpyridyl) porphin has been determined by X-ray crystallography.
The
porphyrin has a precise centre of symnetry; the central core is planar, with
the N-methylpyridyl groups inclined to i t at angles of 66-72°.
Molecular
modelling of t h i s structure into TpA and CpG sites of intercalated DNA, has
been performed, and approximate energetics calculated.
I t has been shown
that only the CpG s i t e can have f u l l ligand i n t e r c a l a t i o n , since the thymine
methyl group s t e r i c a l l y hinders such geometry at TpA s i t e s .
Modelling
indicates the importance of electrostatic effects in the low-energy forms of
intercalated and part-intercalated complexes at both sequences.
INTRODUCTION
The majority of DNA-intercalating molecules incorporate planar aromatic
chromophores with 3-4 fused five or six-membered rings 1 ' . A notable exception
is the porphyrin ligand tetra-(4-N-methylpyridyl) porphyrin (TMPy), together
with several of i t s metal complexes (Fig. 1). The presence of the sterically
bulky N-methylpyridyl groups might be expected to hinder the classical modes of
intercalation as shown by molecules such as proflavine or ethidium; there is
extensive evidence though that TMPy does indeed bind intercalatively to
double-stranded DNA, from for example, unwinding of closed-circular DNA"* .
Several studies have shown that TMPy exhibits sequence-selective DNA
interactions5'6'9'10.
The binding to AT regions has been considered to be
non-intercalative whereas that to GC sequences has been defined as
intercalative. A recent NMR analysis of TMPy interacting with defined-sequence
oligonucleotides has shown11 that C-3'5'-G (CpG) sites are preferred for
intercalation, whilst footprinting experiments 12 " 14 have demonstrated the
existence of prominent binding at both CpG and TpA sites, although this l a t t e r
technique is unable to ascribe these to particular binding modes.
This paper reports on the crystal-structure determination of TMPy i t s e l f ,
together with molecular-modelling and minimum-energy calculations at CpG and
© IR L Press Limited, Oxford, England.
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Fig. 1 . The Structure of TMPy.
TpA s i t e s . We show here that the two sites require distinct binding modes, in
accord with the evidence from solution, and describe the molecular details of
these two categories of TMPy interaction.
EXPERIMENTAL
X-ray Crystallography
TMPy supplied as the tosyiate salt by W.D. Wilson, was crystallised by
the vapour diffusion method from an aqueous solution equilibrated against
methane-pentane 1,2-diol. A deep red crystal of dimensions 0.6x0.2x0.1 mm was
obtained and used for a l l subsequent studies. I t crystallised in the space
group P2 1 /n, with cell dimensions a = 9.346(4)R, b = 12.861(1)A, c =
29.099(10)A and /3= 92.92(3)°. This c e l l , together with the crystal density of
1.26cm , requires that the molecule sits on a crystal lographic centre of
symmetry, with therefore two molecules of TMPy in the unit c e l l .
Intensity
data were measured with an Enraf-Nonius CAD4 diffractometer using an«-20scan
technique, to a l i m i t of 0= 60° using CuKa radiation. A total of 2916 unique
reflections were obtained with / F 0 / < 3 a ( F 0 ) .
Measurement of standard
reflections during the data collection showed no significant decay.
The
structure was solved by direct methods. The positions of hydrogen atoms were
located in subsequent difference Fourier Syntheses, together with two water
molecules of c r y s t a l l i s a t i o n .
These were included in the full-matrix
least-squares refinement process, which converged to a final r e l i a b i l i t y index
R of 0.078. Final non-hydrogen atom positional parameters are given in Table
1.
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TABLE 1 Table
of
Positional Parameters for TMPy
Standard Deviations
Atom
z
0.4218(2)
0.4062(2)
0.5078(2)
0.3947(2)
0.5490(2)
0.3673(2)
0.3971(2)
0.3628 2)
0.3580 2)
0.4948 2)
0.4741(2)
0.4077(2)
0.5403(2)
0.1292(2)
0.3001(3
0.3372(2)
0.4266(2)
0.3583(2)
0.3524(2)
0.3666 3)
0.3922 2)
0.3209 3)
0.3384 3)
0.3068 3)
0.2497 3)
0.255(2)
0.223(2)
X
WT~
CP2
NP1A
CR1
CP12
CP7
CP5
CR4
CP3
CP3A
CP2A
CP6
CP4A
NP1O
CP9
CP8
CP1
CPU
CP4
CR2
CR5
CP1O
CR3
CRO
CR1A
HI
H2
0.0054(5 )
0.0658(7
0.1182(5
0.1931(6
0.1606(6
-0.1973 7
-0.0563(7 i
0.3912(7 i
0.0476(7 i
0.2361 7 I
0.1611(6
-0.1331(6 i
0.2364(7
-0.3230(7 i
-0.1779 9
-0.1157 8
0.1391 6
-0.3453(8
-0.0282(7
0.1057 7
0.3424(7
-0.4032(8
0.1573(8
0.362(1)
-0.388(1)
-0.506(7)
-0.346(7)
and
0.01^5(4)
0.0960(5)
0.1329(4)
0.2602(5)
0.1780(5)
-0.1994(5)
-0.0524(5)
0.3476(6)
0.0756(5)
0.2860(5)
0.1974(5)
-0.1401(5)
0.2739(5)
-0.2991(5)
-0.3026(6)
-0.2521(6)
0.1814(5)
-0.2002(6)
-0.0147(6)
0.3214 6)
0.2758(5)
-0.2500(6)
0.3920(6)
0.4860(9)
-0.3476(8)
-0.358(5)
-0.315(5)
their
Estimated
B(A2)
3io(l)
2.9(1)
2.9(1)
3.2(1)
3.KD
3.KD
3.9(2)
3.5(1)
3.7(2)
3.0(1)
2.9(1)
3.4(1)
4.6(1)
4.7 2)
4.4 2)
2.9 1)
4.4(2)
3.5(1)
4.2
3.4
4.9
5.0
2)
2)
2)
2)
10.4 3)
7.1(2)
Atoms HI and H2 are in the centre of the porphyrin r i n g .
Molecular Modelling
Intercalative interactions of the porphyrin structure as determined by
the
X-ray
crystallographic
study with models
for double-stranded DNA were
evaluated by means of a computer simulation approach analogous to that employed
in e a r l i e r studies in this l a b o r a t o r y 1 5 ' 1 6 .
double-stranded
dCpG sequence
was
taken
analysis of i t s complex with proflavine
dTpA by substitution
of
appropriate
.
The geometry of an intercalated
from the
X-ray
crystallographic
This was altered to the sequence
base atoms.
Energies
of
interaction
between TMPy and intercalation sites was calculated as the sum of electrostatic
and
non-bonded
Lennard-Jones
potentials.
Partial
charges
for
TMPy were
calculated by the CNDO/2 method; those for the dinucleoside monophosphates were
ift
as used previously by u s .
The non-bonded terms were as employed in previous
studies, when they have been found to s a t i s f a c t o r i l y
reproduce experimental
X-ray structures.
Minimum energy configurations for
TMPy in the two intercalation sites
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Fig.
2.
Computer-drawn plot of the molecular
determined crystallographically.
structure
of
TMPy, as
were determined by means of an automated rigid-body docking procedure19. This
systematically explores a l l possible positions with respect to translational
and rotational motions of TMPy within an intercalation s i t e . The conformations
of the dinucleosides were not altered during or subsequent to this process.
All calculations were performed on a VAX 11/750 computer.
Structures
were visualised and examined on Gresham-Lion S214 and Silicon Graphics IRIS
computer graphics systems.
RESULTS
The molecular structure of TMPy, as shown in Figure 2, has a planar
central core, as found in many other porphyrins and 'ruffling' of the core
Table 2 Characteristics of the Intercalation Models for TMPy
Model No.
C
25.8
16.9
1.7
23.4
a.
b.
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E-S
-71.5
-84.2
-76.4
-67.7
L
TOTAL
-97.3
-67.3
-74.7
-91.1
Non-bonded (van der Waals), electrostatic and total energies, are in
kcal mole' 1 . They are on a relative scale.
Electrostatic energy, calculated using a distance-dependent d i e l e c t r i c
constant.
Nucleic Acids Research
3a. Computer-drawn plot of model 1 , f o r the dCpG s i t e and TMPy, viewed
onto the plane of the base p a i r s .
The pyrrole rings of the TMPy
have been shaded.
3b. Plot of model 2 , for the dCpG s i t e .
geometry is not observed here.
this central
The methylpyridyl groups are non-coplanar with
core, and the two independent
rings
of each half-molecule
are
inclined at 65.7° and 71.5° respectively to the core.
Modelling
of
intercalation
of
the
TMPy crystal
structure
into dCpG
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Table 3.
Non-bonded distances between nitrogen atoms of TMPy and
dinucieosides, in A.
Model 1
Model 2
Model 3
Model 4
N1...02P
03'G
5.81
4.95
N2...O2P
03'G
6.50
5.42
N3...01P
05'C
5.39
5.46
N4...01P
05'C
4.99
5.39
N1...01P
05'C
2.91
3.89
N3...01P
05'C
3.35
4.37
N1...01P
05'C
3.73
4.87
N3...05'C
05'C
3.16
3.51
N1...01P
05'C
2.31
3.82
produced two highly discrete minimum-energy positions for the ligand that are
orientated at 45° to each other (Table 2 ) . The more stable one of these, Model
1 , shown in Figure 3a, has a TMPy molecule symmetrically disposed between major
and minor grooves, and thus f u l l y intercalated between base pairs.
The
principle contributions to the stabilisation of this form are electrostatic,
with a l l four pyridyl groups carrying formal cationic charge. These rings are
arranged so that their non-coplanarity with the porphyrin core exerts minimal
interference with the base pairs. There is relatively l i t t l e overlap between
porphin and bases, with the pyrrole rings facing the grooves having least.
Model 2 (Fig.3) represents the only other relatively low-energy configuration
found for TMPy intercalation into dCpG. This i s 30 kcal mole" 1 higher in
energy than model 1, with a substantial non-bonded interaction due to the
proximity of two pyridyl groups to the 05' atoms of the 5'-end deoxyribose
sugars. The more favourable electrostatic term compared to model 1 arises from
the considerably closer contact of these two cationic pyridyl groups to the
phosphate groups, with pyridyl nitrogen
phosphate oxygen atom distances of
2.91A and 3.35A compared to distances of 5.81, 6.50, 5.39 and 4.99A in model 1
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Fig. 4a. Plot of model 3, for the dTpA site.
Fig. 4b. Plot of model 4, for the dTpA site.
(Table 3b). Model 2 has the greater part of the TMPy molecule lying in the
major groove of the ONA sequence; this position has extremely limited vertical
movement (Figure 3 ) , being effectively constrained by the steric hindrance
requirements of the pyridyl rings in relation to the base pairs. The exocyclic
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Fig. 5.
Plot of model 3, rotated by 90° about the horizontal axis
compared to Fig. 4a.
N2 ami no groups of the guanines also play an important role in this constraint,
which forces the TMPy molecule to l i e somewhat asymmetrically with respect to
the horizontal axis of the intercalation site (Fig.3).
The two low-energy models for intercalation into the dTpA duplex sequence
are shown in Figures 4a and 4b. They are models 3 and 4 in Tables 2 and 3.
Both of these are broadly similar to the higher-energy model 2 for the dCpG
sequence. Both have a similar pattern of close contacts that are presumerably
largely electrostatic in nature, between phosphate oxygen atoms and cationic
nitrogen atoms of TMPy. As in model 2, the geometry of these interactions is
remarkably linear in that these oxygen atoms are lying in the Intercalation
plane (Fig.5). The two dTpA-bind1ng modes for TMPy are closely related and
together define the sole low-energy domain for the intercalates in this
sequence, with model 4, the more stable of the two in terms of computed total
energy, having a more asymmetric overall interaction geometry with respect to
the horizontal axis of the dTpA (Fig.4a) Models 3 and 4 share with the dCpG
model 2 the feature of partial intercalation, with the TMPy molecule extending
into the major groove.
The energy scans for the dTpA sequence show that the model 1 symmetric
mode of f u l l intercalation found for dCpG, is of very high relative energy
(>999 kcal mole" 1 of non-bonded energy) for the former sequence. This is
attributable to the presence of the methyl groups on the 5-positions of the
thymines, which are 1n precisely the positions in the TpA major groove that are
occupied by the pyridyl groups of TMPy in model 1 of the dCpG-TMPy complex.
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DISCUSSION
This modelling study has shown that the low-energy structures of the TMPy
intercalation complexes have very distinct geometries at dCpG and dTpA s i t e s ,
and that only the symmetric dCpG model 1 can be considered to be f u l l y
intercalated structure.
I t is of interest that a recent NMR study 11 has
presented evidence in favour of a symmetric dCpG intercalation model, with the
Nl atoms of the guanines close to the central axis of TMPy. The (relatively
small) deviations from this in model 1 are most probably be attributed to the
lack of optimisation of the dinucleoside conformation with respect to the TMPy
molecule. Conversely, the very small or zero imino proton shifts at TpA-bound
TMPy s i t e s 1 0 ' 1 1 are broadly in agreement with the largely non-intercalated
models 3 and 4 presented here, again bearing in mind the lack of refinement in
the model. In general, the AT-site interactions of TMPy have been consistently
interpreted as external ones, on the basis of a large body of experimental
data^" 1 1 .
Whether the dTpA-site models presented here f u l l y reflect these
findings is debatable, since they do involve, at least as presented, an
opening-up of base pairs from 3.4 to 6.8A to form an intercalation s i t e .
However, i t can be shown (unpublished observations), that a wedge-shaped site
that is only slightly opened-up, is possible for a type 4 model.
The
comparability of the electrostatic components to the total energies of models 1
and 4 is a consequence of a l l four pyridyl groups contributing in the former,
whereas in the l a t t e r , there are just two pyridyl groups in rather closer
contact to phosphates. I t is plausible that in the model 1 case, the areas of
space between pyridyl groups and phosphate backbones would be occupied by
solvent, and therefore that the attractive interactions between them would be
substantially diminished; the use here of a distance-dependent dielectric
constant in the calculations cannot adequately compensate for this p o s s i b i l i t y .
We consider i t l i k e l y that the substantially closer (byv3A) distances between
charged groups in the dTpA models, which are too short to have mediated water
molecules between them, indicate in reality a significantly
greater
electrostatic effect in their case, compared to the dCpG one, in accord with
findings on TMPy bound to poly(dA-dT) 9 ' 20 and other ligands externally bound to
DNA1.
The mechanism of TMPy intercalation has been proposed to involve either
an intermediate completely coplanar TMPy structure which would become bound by
classic
intercalation
processes,
or
intercalation
into
transiently
non-base-paired, "breathing" sequences of DNA. The non-coplanarity of the
N-methylpyridyl groups and the porphin core in the crystal structure presented
here, is Indicative of this being a low-energy conformation. Our analysis of
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the energy barrier to rotation suggests that it is sufficiently high for
coplanarity to be effectively discounted, at least without the occurrence of
significant geometric changes to the porphyrin core. We therefore favour the
likelihood of TMPy intercalation being to transiently disrupted ONA sequences,
with consequent slow dissociation kinetics, in agreement with kinetic data in
solution'.
ACKNOWLEDGEMENTS
We are grateful to W.D. Wilson for the provision of material, and
much useful discussion.
T o whom correspondence should be addressed
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