doi:10.1016/S0022-2836(03)00726-5
J. Mol. Biol. (2003) 331, 345–359
Sequence Specific DNA Binding of Ets-1 Transcription
Factor: Molecular Dynamics Study on the Ets
Domain –DNA Complexes
Satoshi Obika, Swarnalatha Y. Reddy and Thomas C. Bruice*
Department of Chemistry and
Biochemistry, University of
California, Santa Barbara, CA
93106, USA
Molecular dynamics (MD) simulations for Ets-1 ETS domain– DNA complexes were performed to investigate the mechanism of sequence-specific
recognition of the GGAA DNA core by the ETS domain. Employing the
crystal structure of the Ets-1 ETS domain– DNA complex as a starting
structure we carried out MD simulations of: (i) the complex between Ets1 ETS domain and a 14 base-pair DNA containing GGAA core sequence
(ETS –GGAA); (ii) the complex between the ETS domain and a DNA having single base-pair mutation, GGAG sequence (ETS – GGAG); and (iii) the
14 base-pair DNA alone (GGAA). Comparative analyses of the MD structures of ETS – GGAA and ETS – GGAG reveal that the DNA bending
angles and the ETS domain– DNA phosphate interactions are similar in
these complexes. These results support that the GGAA core sequence is
distinguished from the mutated GGAG sequence by a direct readout
mechanism in the Ets-1 ETS domain– DNA complex. Further analyses of
the direct contacts in the interface between the helix-3 region of Ets-1 and
the major groove of the core DNA sequence clearly show that the highly
conserved arginine residues, Arg391 and Arg394, play a critical role in
binding to the GGAA core sequence. These arginine residues make bidentate contacts with the nucleobases of GG dinucleotides in GGAA core
sequence. In ETS – GGAA, the hydroxyl group of Tyr395 is hydrogen
bonded to N7 nitrogen of A3 (the third adenosine in the GGAA core),
while the hydroxyl group makes a contact with N4 nitrogen of C4 (the
complementary nucleotide of the fourth guanosine G4 in the GGAG
sequence) in the ETS –GGAG complex. We have found that this difference
in behavior of Tyr395 results in the relatively large motion of helix-3 in the
ETS – GGAG complex, causing the collapse of bidentate contacts between
Arg391/Arg394 and the GG dinucleotides in the GGAG sequence.
0
q 2003 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: molecular dynamics; Ets-1; ETS domain; transcription factor;
protein –DNA complex
Introduction
The ETS protein family contains more than 45
eukaryotic transcription activators and inhibitors,
such as Ets-1, PU.1, Fli-1, GABPa, SAP-1, TEL and
Elk-1.1 – 3 Members of this family play an important
role in normal cell proliferation and differentiation.
The DNA rearrangement and/or overexpression of
ets gene have been known to lead to
tumorigenesis.4 In order to regulate gene
expression, the ETS family of proteins bind to a
Abbreviation used: MD, molecular dynamics.
E-mail address of the corresponding author:
[email protected]
consensus DNA sequence centered on the core
sequence 50 -GGA(A/T)-30 through the highly conserved DNA-binding domain.3 The DNA-binding
domain for ETS proteins, termed ETS domain, is
about 85 amino acid residues in length and forms
a winged helix-turn-helix motif consisting of three
a-helices and four b-strands. The recent X-ray5 – 10
and NMR11 – 13 studies of the ETS domain –DNA
complexes have shown that the helix-3 in the
winged helix-turn-helix motif binds in the major
groove of the consensus DNA sequence.
In the crystal structure of Ets-1 ETS domain –
DNA [d(TAGTGCCGGAAATGT)2] complex (PDB
code: 1K79), two arginine residues, Arg391 and
Arg394, which are in the helix-3 region and
0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
346
MD Simulations of Ets-1 ETS Domain–DNA Complexes
conserved among the ETS family, make bidentate
interactions with G1 and G2, respectively (Figure
1).5 However, the pattern of these hydrogen bonds
is not maintained in the crystal structures of other
ETS domain– DNA complexes.7,9,10 In addition, the
interaction between the arginine residues, Arg391
and Arg394, and the consensus DNA sequence is
not observed in the NMR study on Ets-1 ETS
domain– DNA complex.12,13
On the other hand, a few studies have proposed
direct contacts of the amino acid residues in ETS
domain with the AA region (þ 3 and þ 4 positions)
in the GGAA core. For example, an X-ray study of
the Ets-1 ETS domain –DNA complex indicated
what would be a vital role of hydrophobic interaction between the phenyl ring of Tyr395 and 5methyl group of T4 or T5 .5 However, this type of
interaction was not observed in other ETS
domain– DNA complexes,6 nor is the tyrosine residue conserved in other ETS family proteins such
as PU.1 and TEL.9,10 Thus, the precise molecular
mechanism that clearly explains the sequencespecific GGAA recognition by ETS domain is still
lacking.
The phosphate groups of DNA adjacent to the
core sequence GGAA have contacts to the winged
segment and the turn region between helix-2 and
helix-3 of the ETS domain. It was reported that the
neutralization of anionic phosphate charges on
one face of DNA resulted in the DNA bending,
probably due to the electrostatic repulsions of the
remaining anionic charges.14 – 16 In fact, DNA bending was observed in the crystal structures of the
ETS domain –DNA complexes.5 – 10 It was also supposed that the conformational change of DNA
caused by the DNA bending would serve to provide effective GGAA core recognition by the helix3 of the ETS domain. However, the bending angles
of DNA previously reported in the X-ray crystallographic analyses of ETS domain– DNA complexes
vary from one system to another.5 – 10 Thus,
additional investigation is required in order to clarify a common role of the DNA bending in the
sequence-specific binding of the ETS domain.
Significant developments have been made in the
last few years in the procedures of molecular
dynamics (MD). Improvements in AMBER,17
CHARMM18,19 and GROMOS20 force fields and
effective treatment of long-range electrostatic interactions by using particle mesh Ewald (PME)
method,21 explicit inclusion of solvent and ions
have opened the possibilities for accurate determination of protein and DNA structures.22 Besides
availability of supercomputers have enabled to
undertake simulations on a nanosecond (ns) timescale which expanded conformational sampling
and eventually to elucidate biomolecular interactions. Further understanding of the molecular
mechanism of sequence-specific DNA binding of
the ETS domain is likely to provide novel clues for
the design of drugs that bind to inhibit the interaction between the ETS domain and DNA. We
report here 3.5– 3.9 ns MD simulations of two Ets1 ETS domain –DNA complex systems. The amino
acid sequence of Ets-1 ETS domain and the DNA
sequences used in this study are shown in Figure 2.
The binding activity of the ETS domain is known
to be higher than that of the whole ETS-1
protein.23 – 27 Therefore, the ETS domain– DNA
complex would be a good model system for MD
simulation. The first system has the ETS domain
(103 amino acid residues) of Ets-1 protein (Figure
2(a)) and the high affinity 14 base-pair DNA containing GGAA core (þ 1 to þ 4, Figure 2(b))
sequence, while the second one has a low affinity
DNA involving a mutation of a single base-pair,
GGAG sequence. In the text, we refer to these complexes as ETS – GGAA and ETS – GGAG, respectively. In addition, results from the MD simulation
of the 14 base-pair DNA having the GGAA core
sequence (referred as GGAA) are also discussed
for comparison.
0
0
Figure 1. Crystal structure of Ets-1 ETS domain – DNA complex (PDB code: 1K79): (a) the whole structure, (b) the
close-up view of the ETS domain – DNA nucleobase contact site (helix-3 and GGAA core sequence).
347
MD Simulations of Ets-1 ETS Domain–DNA Complexes
Figure 2. Sequences of Ets-1 ETS
domain and 14 base-pair DNA: (a)
amino acid sequence with the residue numbers and secondary structure indicated above the sequence,
(b) DNA base sequence with the
numbering provided above or
below the sequence. Residues in
the core GGAA are shown in red.
In the GGAG sequence, the
mutated GC base-pair is shown in
blue.
Results
The root-mean-square deviations (RMSD) of the
protein backbone and DNA heavy-atoms with
respect to the minimized structures of ETS
domain– DNA complexes and DNA (GGAA) are
given in Figure 3. During the simulation, the
RMSD values of the protein fluctuated around
1.16 –2.12 Å in ETS –GGAA and 0.92– 1.55 Å in
ETS –GGAG (Figure 3(a)), while those of DNA in
ETS –GGAA and ETS – GGAG are from 1.06 Å to
2.18 Å and from 1.12 Å to 1.96 Å, respectively
(Figure 3(b)). The plots indicate the stability at
about 900 ps, except in the ETS –GGAA protein
structure. The DNA structure of GGAA exhibits
relatively large RMSD values, compared to the
ETS –GGAA and ETS –GGAG complexes (green
line in Figure 3(b)). This indicates the absence of
ETS domain would cause conformational changes
in the DNA structure.
The positional fluctuations of Ca atoms (CA) of
Ets-1 ETS domain evaluated from MD trajectories
are shown in Figure 4 along with that from the
crystal structure. Although the magnitude of the
fluctuations from X-ray and MD structures are
different, the fluctuation pattern is similar. The
helix-3 region (residues 386 – 396) of the ETS
domain that recognizes the core DNA sequence
has smaller fluctuations, compared to the other
part of the protein. On the other hand, the turn
region (379 – 384) between helix-2 and helix-3 and
the winged region (405 –410) show larger fluctu-
ations. The steep peak observed in the C-terminal
of helix-1 (348 – 353) of ETS – GGAA MD structure
is likely due to the contact between the residues
348– 353 and helix-5 region (430 – 436). However, it
does not seem to affect any other structural features of the ETS domain.
DNA structure of Ets-1 ETS
domain –DNA complex
DNA bending
The time-variation plots of DNA bending angle
for the ETS – GGAA and ETS – GGAG MD structures are given in Figure 5(a). According to the
literature,14,28 the DNA bending angle is defined as
shown in Figure 5(b). Large fluctuations are
noticed in the bending angle with average value
of 168 and 228 for ETS – GGAA (900 – 3480 ps) and
ETS – GGAG (900 – 3930 ps), respectively. In GGAA
helix, a relatively extended DNA structure is
obtained compared to the ETS –DNA complexes.
The average (900 –3255 ps) bending angle of
GGAA is found to be 118. A stereo plot of average
DNA structures of ETS –GGAA (900 – 3480 ps),
ETS – GGAG (900 – 3930 ps) and GGAA (900 –
3255 ps) are given in Figure 5(c) – (e), superimposed
on the canonical B-DNA structure.29 The plots indicate that the presence of ETS domain influences the
DNA bending. As can be seen in Figure 5(a), (c),
and (d), no significant difference in DNA bending
348
MD Simulations of Ets-1 ETS Domain–DNA Complexes
is observed in the ETS –GGAA and ETS –GGAG
complexes.
Major and minor groove widths
Figure 3. Time evolution of RMSD of the MD complexes of ETS– GGAA (black) and ETS– GGAG (red)
with respect to the corresponding minimized structures:
(a) the backbone heavy-atoms of the ETS domain and
(b) all the heavy-atoms of DNA except terminal basepairs. The RMSD value of the heavy-atoms of GGAA
helix (green) with respect to minimized structure is
shown in (b).
According to the literature,14 groove widths are
defined as distances between two appropriate
phosphate atoms (see Method). The average major
and minor groove widths of 14 base-pair DNA in
ETS –GGAA, ETS – GGAG and GGAA are summarized in Table 1. The major groove width around the
core GGAA region is found to be comparatively
large in the crystal structure (PDB code: 1K79),
although the DNA bends into the major groove. In
the MD averaged structure, an expansion of major
groove width around the core region is also
observed. For example, the averaged major groove
widths at base-pair 1 in ETS –GGAA and GGAA
are 20.43 Å and 17.11 Å, respectively. Thus, the
MD simulation of the ETS domain– DNA complex
reproduced this structural feature well. Noteworthy are the observations that fluctuations in
the major groove width around the core region
(base-pairs 1– 4) are quite small in ETS – GGAA,
while large fluctuations are observed in the major
groove width at the base-pairs 3 and 4 in ETS –
GGAG complex. These small fluctuations in the
major groove width of ETS – GGAA may reflect
the stability of the interaction between the helix-3
of ETS domain and the GGAA core sequence.
In comparison to the dynamics of the major
groove, the minor groove widths obtained from
MD simulations of ETS –GGAA, ETS – GGAG and
GGAA show little difference between each other.
The values of minor groove width in the crystal
Table 1. Average major and minor groove widths (Å)
and their standard deviations (in parenthesis) of the 14
base-pair DNA duplexes of ETS– GGAA, ETS– GGAG
and GGAA MD structures. The values of X-ray structure
of ETS– GGAA (PDB code: 1K79) are given
ETS–GGAA
(900–
3480 ps)
ETS–GGAG
(900–
3930 ps)
GGAA
(900–
3255 ps)
Major groove
23
18.71
22
17.90
21
17.75
1
20.47
2
19.87
3
18.45
4
17.06
5
18.82
6
18.90
17.91(^1.50)
17.91(^1.64)
18.56(^1.17)
20.43(^0.84)
20.82(^0.43)
20.40(^0.85)
18.22(^0.75)
17.94(^1.00)
18.61(^1.54)
17.55(^1.64)
18.29(^1.67)
18.13(^1.15)
19.33(^0.80)
20.52(^0.40)
19.10(^1.63)
18.97(^1.79)
19.96(^1.90)
19.92(^1.85)
17.00(^1.49)
17.54(^1.71)
17.32(^1.55)
17.11(^1.93)
17.41(^1.78)
16.80(^1.50)
16.82(^1.24)
15.90(^1.46)
16.80(^1.40)
Minor groove
23
13.88
22
13.52
21
14.90
1
13.46
2
14.14
3
13.21
4
9.98
5
9.06
14.20(^0.88)
13.77(^1.27)
15.48(^1.13)
14.96(^1.05)
14.60(^1.23)
13.80(^1.24)
12.67(^1.34)
12.02(^1.21)
13.86(^1.09)
14.74(^1.21)
15.81(^1.57)
15.60(^1.28)
14.68(^1.70)
14.83(^1.33)
13.54(^1.05)
11.11(^1.17)
13.70(^1.16)
14.11(^1.39)
14.57(^1.17)
14.52(^1.14)
13.84(^1.12)
13.22(^0.88)
13.11(^1.02)
13.40(^1.02)
Basepair
Figure 4. Atomic fluctuations of Ca atoms of the Ets-1
ETS domain in MD structures of ETS – GGAA (averaged
for 900– 3480 ps) in black, and ETS – GGAG (averaged
for 900–3930 ps) in red. Fluctuations of Ca atom of the
crystal structure are shown in blue.
X-ray
structure
(PDB:
1K79)
349
MD Simulations of Ets-1 ETS Domain–DNA Complexes
Figure 5. (a) Time evolution of
the DNA bending angle u (deg) in
the MD structures of ETS – GGAA
(black) and ETS– GGAG (red). The
bending angle of the crystal structure is shown in blue. (b) Schematic
representation of definition of DNA
bending angle u. Stereo view of the
averaged DNA structures obtained
from the MD simulations of (c)
ETS– GGAA
(900 – 3480 ps)
in
black, (d) ETS –GGAG (900 –
3930 ps) in red and (e) GGAA
(900 – 3255 ps) in green. The canonical B-form DNA structure is also
shown (blue) in (c) – (e) for comparison. Superimpositioning is performed
according
to
the
orientations of base-pairs 24, 25
and 26. All hydrogen atoms are
omitted for clarification.
structure are consistent with those in the MD structure of ETS – GGAA, except for the values at basepairs 4 and 5.
Sugar puckering
The time evolution of pseudorotational phase
angles ðPÞ of selected nucleosides in both ETS
domain– DNA complexes are represented in
Figure 6. The switching of sugar puckering from
C2 -endo (S-form) to C3 -endo (N-form) or from Nform to S-form is observed in some nucleoside residues (Figure 6(b), (c), and (e)). On the other hand,
the flexibility in sugar puckering is restricted in
some DNA regions where the phosphates or
nucleobases have contacts with the ETS domain.
0
0
Especially, the sugar conformations of G23 and A6
are found to be almost locked in S-form puckering
(Figure 6(a) and (f)) due to their 30 -phosphate
group being rigidly held in an interaction with
specific amino acid residues in the ETS domain.
The 30 -phosphate oxygen atoms, O1P and O2P of
G23 (by convention listed in Table 2 as the 50 -phosphate oxygen atoms of the neighboring C22,
O1P(C22) and O2P(C22), respectively) contact with
the hydroxyl oxygen OH of Tyr386, side-chain
amino nitrogen NZ of Lys404 and main-chain
amino nitrogen N of Tyr410, while those of A6
(listed as O1P(T5 ) and O2P(T5 ) form a salt bridge
with N(Leu337) and OH(Tyr396). A considerable
difference in the puckering between the ETS –
GGAA and ETS – GGAG structures is observed at
0
0
0
0
350
MD Simulations of Ets-1 ETS Domain–DNA Complexes
Figure 6. Time evolution of the pseudorotation phase angle P of sugar ring of nucleosides (a) G23, (b) C23 , (c) C21, (d)
G2, (e) C2 and (f) A6 in the MD structures of ETS– GGAA (black) and ETS– GGAG (red).
0
0
0
C21 (Figure 6(c)). This nucleoside residue in ETS –
GGAA complex likely prefers the S-type sugar conformation, while the sugar puckering of C21 in
ETS –GGAG remains N-type after 2500 ps.
Interaction between Ets-1 ETS domain and DNA
Contacts of Arg391 and Arg394 with nucleobases
in the core sequence
The distances between non-bonded entities in
the contact region of the ETS domain– DNA complexes determined by MD simulation and crystal
structures are summarized in Table 3. The hydrogen bonding structures involving Arg391 and
Arg394 with nucleobases are shown in Figure 7.
The time variations of selected heavy-atom nonbonded distances are shown in Figure 8. In the
ETS –GGAA MD structure, Arg391 of ETS is in
bidentate contacts with G2 nucleobase by hydrogen
bonds between secondary amino nitrogen NE of
Arg391 and O6 oxygen of G2, and the other
between guanido nitrogen NH2 of Arg391 and N7
nitrogen of G2 (Figure 7(a)). These two hydrogen
bonds are maintained during the entire simulation
(black line of Figure 8(a) and (c)). Such hydrogen
bond interactions are observed between Arg394
and G1 nucleobase (Figure 7(b)) for the duration
after 1700 ps in ETS – GGAA (black line of Figure
8(d) and (f)). These separations are large for the
Table 2. Average non-bonded distances (Å) and their standard deviations (in parenthesis) of the contact sites of Ets-1 –
DNA phosphate backbone of ETS – GGAA and ETS– GGAG MD structures. The values of X-ray structure of ETS –
GGAA (PDB code: 1K79) are given
ETS–GGAA
Atoms
N(Leu337)· · ·O1P(T5 )
NE1(Trp375)· · ·O2P(T4 )
NZ(Lys379)· · ·O1P(T4 )
OH(Tyr386)· · ·O2P(C22)
NZ(Lys388)· · ·O2P(T3 )
OH(Tyr396)· · ·O2P(T5 )
OH(Tyr397)· · ·O2P(C21)
NZ(Lys399)· · ·O2P(A6 )
NZ(Lys404)· · ·O1P(C22)
N(Tyr410)· · ·O1P(C22)
OH(Tyr410)· · ·O1P(G23)
0
0
0
0
0
0
ETS–GGAG
X-ray structure (PDB: 1K79)
(900–1650 ps)
(1800–3480 ps)
(900– 2100 ps)
(2700–3930 ps)
2.80
2.86
2.85
2.52
2.95
2.46
2.66
2.64
2.42
2.83
2.58
2.94(^0.20)*
3.53(^0.50)
4.87(^1.18)
2.65(^0.10)*
4.71(^0.32)
2.64(^0.10)*
2.74(^0.14)*
3.79(^1.21)
2.72(^0.11)**
2.83(^0.13)*
2.86(^0.48)**
2.96(^0.27)*
3.22(^0.51)**
5.35(^0.67)
2.65(^0.10)*
2.79(^0.33)*
2.81(^0.37)**
2.72(^0.13)*
3.72(^0.93)
2.74(^0.12)**
2.87(^0.15)*
3.03(^0.69)**
3.00(^0.29)*
2.96(^0.31)*
2.77(^0.18)*
2.65(^0.10)*
2.75(^0.13)*
2.94(^0.49)**
2.71(^0.12)*
3.98(^0.94)
2.87(^0.33)**
3.01(^0.28)*
2.66(^0.15)*
2.96(^0.18)*
3.02(^0.40)*
2.73(^0.14)*
2.65(^0.10)*
2.70(^0.10)*
2.64(^0.10)*
2.69(^0.12)*
3.38(^0.83)
2.73(^0.11)**
3.00(^0.21)*
2.64(^0.10)*
Values within *0–0.25 Å and **0.25–0.50 Å different from the X-ray structure.
351
MD Simulations of Ets-1 ETS Domain–DNA Complexes
Table 3. Average non-bonded distances (Å) and their standard deviations (in parenthesis) of the contact sites of Ets-1 –
DNA nucleobase of ETS– GGAA and ETS – GGAG MD structures. The values of X-ray structure of ETS – GGAA (PDB
code: 1K79) are given
ETS –GGAA
Atoms
NE(Arg391)· · ·O6(G2)
NH2(Arg391)· · ·O6(G2)
NH2(Arg391)· · ·N7(G2)
NE(Arg394)· · ·N7(G1)
NH2(Arg394)· · ·N7(G1)
NH2(Arg394)· · ·O6(G1)
OH(Tyr395)· · ·N6(A3)
OH(Tyr395)· · ·N6/O6(A4/G4)
OH(Tyr395)· · ·O4/N4(T4 /C4 )
CD2(Tyr395)· · ·C5M(T5 )
CE2(Tyr395)· · ·C5M(T5 )
0
0
0
0
ETS –GGAG
X-ray structure (PDB: 1K79)
(900 –1650 ps)
(1800–3480 ps)
(900–2100 ps)
(2700– 3930 ps)
2.65
3.67
2.77
2.84
3.67
2.85
3.10
3.64
3.77
3.41
3.42
2.90(^0.15)*
3.94(^0.29)**
2.90(^0.10)*
3.96(^0.53)
3.04(^0.21)
3.97(^0.62)
3.42(^0.38)**
3.98(^0.60)**
3.52(^0.55)*
3.96(^0.28)
3.82(^0.30)**
2.96(^0.19)**
4.20(^0.35)
2.90(^0.13)*
2.97(^0.12)*
3.58(^0.28)*
2.91(^0.17)*
3.09(^0.26)*
3.63(^0.40)*
3.58(^0.51)*
3.92(^0.38)
4.05(^0.43)
2.97(^0.17)**
4.19(^0.31)
2.89(^0.10)*
2.94(^0.13)*
3.67(^0.30)*
2.91(^0.17)*
3.29(^0.27)*
4.36(^0.38)
3.08(^0.25)
3.97(^0.37)
3.87(^0.31)**
3.91(^0.46)
2.97(^0.33)
3.00(^0.15)*
5.16(^0.17)
3.00(^0.13)
5.13(^0.20)
4.27(^0.63)
4.64(^0.49)
3.00(^0.18)
3.93(^0.34)
3.75(^0.27)**
*
Values within 0–0.25 Å and **0.25–0.50 Å different from the X-ray structure.
time-period 250– 1700 ps, during which a hydrogen bond interaction is observed between the guanido nitrogen NH2 of Arg394 and N7 nitrogen of
G1 nucleobase (black line of Figure 8(e)).
In the case of ETS – GGAG, the contact between
NE of Arg391 and O6 of G2 (red line of Figure
8(a)) give way to a hydrogen bond between NH2
of Arg391 and O6 of G2 during 2850– 2900 ps
(Figure 7(c) and red line of Figure 8(b)). Although
NH2 of Arg391 is within 3.0 Å distance from N7
nitrogen of G2 even after 2850 ps, the non-bonded
angle for NH2(Arg391)– HH21(Arg391)· · ·N7(G2)
is 120.7(^ 27.5)8. This does not allow hydrogen
bond formation (Figure 7(c)).30 Arg394 has a contact with G1 forming two hydrogen bonds one
between NE of Arg394 and N7 nitrogen of G1 and
the other between NH2 of Arg394 and O6 oxygen
of G1. These interactions exist until 2100 ps of
dynamics (red line of Figure 8(d) and (f)). The contact between NH1 of Arg394 and OG of Ser390,
which would stabilize the helix-3 structure, is also
broken during the same time (data not shown). In
place, NH2 of Arg394 forms a hydrogen bond
with N7 nitrogen of G1 (Figures 7(d) and 8(e)). So
Figure 7. Molecular plot showing the contact interactions of MD structures. (a) Arg391 with G2C2 DNA base-pair
and (b) Arg394 with G1C1 base-pair in ETS – GGAA, averaged for 1800 –3480 ps; (c) Arg391 with G2C2 base-pair and
(d) Arg394 with G1C1 base-pair in ETS – GGAG, averaged for 2700– 3930 ps. See the stable bidentate hydrogen bonds
in (a) and (b), while they are absent in (c) and (d).
0
0
0
0
352
MD Simulations of Ets-1 ETS Domain–DNA Complexes
Figure 8. Time-dependent variation of separations of (a)
NE(Arg391)· · ·O6(G2),
(b) NH2(Arg391)· · ·O6(G2),
(c) NH2(Arg391)· · ·N7(G2),
(d) NE(Arg394)· · ·N7(G1),
(e) NH2(Arg394)· · ·N7(G1) and
(f) NH2(Arg394)· · ·O6(G1) of ETS –
GGAA (black) and ETS– GGAG
(red) MD structures.
during the course of dynamics certain structural
alignments prevail favoring some interactions at
the cost of others.
In the crystal structure of Ets-1 ETS domain–
DNA complex, Tyr395 is proximal to A4 and T4
nucleobases in the major groove of the core region
(Figure 1).5 As shown in Table 3, the hydroxyl
group of Tyr395 is at 3.09 and 3.63 Å from the exocyclic amino nitrogen atoms N6 of A3 and A4,
respectively in the MD structure of ETS – GGAA.
This indicates that the hydroxyl group forms a
hydrogen bond with N6 of A3, while it makes a
weak contact at A4. The interaction between the
hydroxyl group of Tyr395 and the carbonyl oxygen
O4 of T4 is also observed. However, the close contact, in which the distance between the hydroxyl
group of Tyr395 and O4 of T4 is less than 3.2 Å, is
identified only for short time-periods (1000 –
1200 ps and 2250 – 2600 ps). The steric hindrance
caused by the 5-methyl group of T4 likely prevents
hydrogen bonding between the O4 carbonyl oxygen of T4 and the hydroxyl group of Tyr395.
During dynamics the delta carbon CD2 of Tyr395
is at 3.92 Å from the 5-methyl carbon C5M of T5 ,
indicating hydrophobic interaction between the
phenyl ring of Tyr395 and the 5-methyl group of
T5 (Table 3).
In the MD structure of ETS – GGAG, a contact
between the hydroxyl group of Tyr395 and N6
atom of A3 is observed until about 2800 ps
(Table 3). The Tyr395 hydroxyl group forms a
hydrogen bond with the 4-amino nitrogen N4 of
C4 nucleobase. Unlike the ETS – GGAA complex
where the 5-methyl group of T4 prevents hydrogen
bonding, the C4 in ETS – GGAG is in contact with
Tyr395. The hydrophobic interaction between C5M
0
0
0
0
0
0
0
0
0
Motion of the helix-3 on the interface between
ETS domain and DNA
Contact of Tyr395 with the DNA
0
of T5 and the phenyl ring of Tyr395 is also seen in
the ETS – GGAG complex.
In order to investigate the motion of the helix-3
in the major groove of the DNA, the structures
were averaged at the intervals of 300 ps and analyzed. Some structures are superimposed according to the orientations of G2, A3 and A4/G4 (Figure
9). The stereo pictures indicate that the position
and motion of the helix-3 in each complex are
quite different. In the ETS –GGAA complex, the
helix-3 is settled in the major groove of the consensus DNA sequence without significant positional
fluctuations. The main-chain of the helix-3 (CA, C
and N atoms) is almost at the same position during
the entire duration of MD simulation (Figure 9(a)),
and the contact residues, Arg391, Arg394 and
Tyr395 show no change in conformation (Figure
9(b)). In addition, overall the helix-3 region is similar in both X-ray and MD averaged structures (data
not shown).
On the contrary, a distinct motion of the helix-3
region can be seen in the ETS – GGAG complex
(Figure 9(c) and (d)). The main-chain of the helix-3
gradually moves as the simulation proceeds. For
example, the Ca atom of Tyr395 in the MD structure averaged for 1200 –1500 ps (yellow structure
in Figure 9(d)) shows a movement of 4.11 Å with
respect to the MD structure averaged for 3000 –
3300 ps (blue structure in Figure 9(d)).
Interaction between the ETS domain and
phosphate backbone in the DNA
Eleven direct contacts between hydrogen donors
in the ETS domain and phosphate backbone in the
353
MD Simulations of Ets-1 ETS Domain–DNA Complexes
Figure 9. Stereo diagrams of the helix-3 in the major groove of core DNA sequence in (a),(b) ETS– GGAA and (c),(d)
ETS– GGAG MD structure. The MD structures averaged for the period 1200– 1500 ps (yellow), 1800– 2100 ps (green),
2400– 2700 ps (gray) and 3000– 3300 ps (blue) are superimposed according to the orientations of G2, A3 and A4/G4. In
(a) and (c), the backbone atoms (CA, C and N) of binding site (residues 386– 396) corresponds to the major groove
view of DNA (base-pairs 1 –5). In (b) and (d), the close-view of (a) and (c) perpendicular to the helix axis are given,
with only few important protein residues (Arg391, Arg394 and Tyr395) and nucleobases (G1, G2, A3, A4/G4, T4 and
T5 ) shown.
0
0
354
MD Simulations of Ets-1 ETS Domain–DNA Complexes
DNA are observed in the crystal structure of the
ETS –GGAA complex (PDB code: 1K79).5 These
are listed in Table 2, along with the corresponding
distances of ETS – DNA MD complexes. Only slight
differences (, 0.5 Å) in the contact distances of salt
bridge formations between the ETS domain and
phosphate backbone of DNA are observed
between the crystal and the averaged MD structures. In ETS –GGAA, the contacts between the primary amino nitrogen NZ of Lys379 and the
phosphate oxygen O1P of T4 and between NZ of
Lys399 and O2P of A6 are not seen.
As seen in Figure 8, the drastic changes are
observed in the hydrogen bonding pattern
between DNA base and the helix-3 region of the
ETS –GGAG MD structure. However, no considerable change in the direct contact of the turn and
winged region of the ETS domain (two ends of the
helix-3 region) with the DNA phosphate backbone
is noticed in the MD averaged structures (900 –
2100 and 2700– 3930 ps) of ETS –GGAG. This result
suggests the possibility that the helix-3 works independently of the flanked regions in the ETS
domain– DNA interaction.
in the bending angle in the range 5– 398 and 5 –
428, respectively (Figure 5(a)). These results indicate that the differences in the DNA bending
angle are likely to arise due to flexibility of DNA
helix in the complexes.
Analysis of Ets-1 ETS domain– DNA complex in
X-ray structures has led to the proposal that the
reduction in binding affinity of ETS –GGAG complex is due to the absence of van der Waals contacts with C4 , and the reduction in the van der
Waals overlap between the 5-methyl group of T5
and the phenyl ring of Tyr395.5 The possibility of
hydrogen bond formation between the hydroxyl
group in Tyr395 and N4 of C4 was also reported
in the literature.5 However, it was concluded that
the contact would not significantly affect the binding energy. These explanations from X-ray analysis
are not consistent with the MD simulations. The
distance between the 5-methyl group of T5 and
the delta carbon CD2 of Tyr395 observed in the
MD structure of ETS –GGAA are comparable to
that of ETS – GGAG (Table 3), indicating that the
van der Waals interaction between the 5-methyl
group of T5 and the phenyl ring of Tyr395 does
not play an important role in the recognition of
the GGAA core sequence. In addition, the hydroxyl
group of Tyr395 is found to be hydrogen bonded to
N4 of C4 in the MD simulation of ETS –GGAG and
is maintained during the entire simulation. However, in ETS – GGAA the contact of the hydroxyl
group with O4 of T4 is only observed intermittently. These results indicate the significance of the
contact between the hydroxyl group of Tyr395 and
N4 of C4 . It is noteworthy that the specific recognition of the nucleobase 40 -position by ETS domain
is observed in ETS –GGAG, but not in ETS – GGAA.
0
Discussion
0
0
0
0
0
0
Comparison of the MD structures to the
experimental data
0
MD simulations on ETS – GGAA and ETS –
GGAG clearly show the presence of meta-stable
states of hydrogen bonding in which the conserved
residues, Arg391 and Arg394, are participating
(Figure 7). In the low affinity ETS – GGAG complex,
both arginine residues change hydrogen bonding
partners after 2 ns MD simulation. In contrast,
Arg394 in ETS –GGAA changes only its side-chain
conformation preference at around 1.7 ns to form
more stable bidentate hydrogen bonds with the
same partner (Figure 8(d) and (f)). Thus, the arginine residues show a certain degree of conformational flexibility in the ETS domain– DNA
complexes. These observations agree well with the
results from NMR experiments of Ets-112,13 and
Fli-1,11 in which the conserved arginine residues in
the ETS domain– DNA complexes were not
assigned, and it was concluded that the Arg391
and Arg394 did not have a single defined conformation in the complexes. Furthermore, the hydrogen bonding mode of these arginine residues in
the crystal structures of ETS domain –DNA complexes depends on the complex studied,5 – 10 which
also supports the conformational flexibility of the
arginine residues.
In the crystal structures of ETS domain– DNA
complexes, the DNA bending angle varies from
structure to structure.5 – 10 The X-ray structure of
PU.1 –DNA complex shows a DNA bending angle
of 88,9,10 while the value in the Ets-1 –DNA complex
was reported5 to be 278. MD studies on ETS –
GGAA and ETS –GGAG show major fluctuations
0
Direct and indirect readout mechanism
X-ray crystallographic analysis of protein– DNA
complexes often reveals a distorted structure of
DNA helix comprising a bending and a kinking
structure.31,32 In some of these cases, it seems to be
difficult to explain all the sequence-specificity
based only by the direct and water-mediated interactions between the protein and such a distorted
DNA. An indirect readout mechanism has been
proposed to explain the sequence-specific DNA
binding of protein.33 – 37 In the indirect readout
mechanism, a protein recognizes a sequencedependent DNA conformation that already exists
before binding or, alternatively, is induced after
binding. A comparative analysis of the DNA binding specificity of other ETS family proteins (Fli-1,
SAP-1, PU.1 and TEL) using a multiplex and other
experimental techniques was reported, wherein a
possibility of the indirect readout mechanism in
recognition of GGA core flanking regions (2 3,
2 2, 2 1, 4, 5 and 6 base-pairs) was mentioned.38
However, no significant difference in either DNA
bending angle (Figure 5(a), (d), and (e)) and ETS
domain–DNA phosphates interactions (Table 3) are
observed between ETS–GGAA and ETS–GGAG
MD Simulations of Ets-1 ETS Domain–DNA Complexes
355
during MD simulations. This is sufficient to
explain an advanced stability of the ETS –GGAA
complex. These results suggest that the AT basepair at position þ 4 is recognized by the direct
readout mechanism, but not by the indirect readout mechanism in the Ets-1 ETS domain–DNA
complex.
form stable hydrogen bonds in the MD structure
(Figure 9). Unlike the ETS – GGAG complex, the
hydroxyl group of Tyr395 does not have a stable
interaction with O4 of T4 due to a steric hindrance
by the 5-methyl group of T4 . Instead, the hydroxyl
group makes a contact with N6 of A3. Consequently, the position of Tyr395 and the helix-3
does not change during the MD simulation, allowing a stable interaction between the helix-3 and
the GGAA core sequence. Thus, the Tyr395 might
work as a tactile sensor to distinguish a targeted
AT base-pair at the þ 4 position.
Role of Arg391, Arg394 and Tyr395 in
recognition of the core DNA sequence
The MD studies on the ETS –GGAA and ETS –
GGAG complexes clearly show that the highly conserved Arg391 and Arg394 play an important role
in the interaction between the helix-3 and the
major groove of GGAA core sequence (Figures 7
and 8) consistent to X-ray studies of ETS domain –
DNA complexes.5 – 10 The two bidentate hydrogen
bonds formed between Arg391 and G2, and
between Arg391 and G1 are stable in the MD simulation of ETS – GGAA, while the corresponding
interactions are not maintained in the simulation
of ETS –GGAG. The decrease in the stability of the
ETS –GGAG complex should be due to the collapse
of these bidentate interactions. It is of great interest
that the two arginine residues, Arg391 and Arg394,
show such a different behavior in the high and low
affinity complexes, though they contact the conserved GG sequence (þ 1 and þ 2 positions) in
each complex. These arginine residues are highly
conserved among the ETS protein family and are
well known to take part in DNA binding. Thus,
the results obtained here are likely to be meaningful for a clear understanding of the sequencespecific DNA binding of the ETS domain.
The Tyr395 residue of the ETS domain plays a
critical role in the recognition of the GG sequence
by the conserved arginine residues, Arg391 and
Arg394. Tyr395 is a neighboring residue of
Arg394. Therefore, the motion of Tyr395 should
have a direct influence on the location or movement of Arg394. In the MD structure of ETS –
GGAG, the hydroxyl group of Tyr395 is hydrogen
bonded with N4 of C4 at the beginning of simulation. This hydrogen bond formation resulted, to
some extent, in the motion of Tyr395 in the major
groove of the GGAG sequence. The Arg394 is
directly affected by the motion of Tyr395 and the
bidentate hydrogen bonds between Arg394 and G1
are lost at 2100 ps (Figure 8(a) and (c)). Thereafter,
a large amount of motion of the helix-3 in the
major groove of GGAG sequence is observed
(gray structure of Figure 9(c) and (d)). As a result
of these motions, the bidentate interactions
between the Arg391 and G2 are absent after
2850 ps. These conformational changes are related
to each other and arise from hydrogen bond formation between the hydroxyl group of Tyr395 and
N4 of C4 .
On the contrary, the helix-3 region is localized in
the major groove of the core DNA sequence of
ETS –GGAA without considerable fluctuations,
and consequently the conserved arginine residues
0
0
0
0
Conclusions
We conducted 3.5 –3.9 ns MD simulations of Ets1 ETS domain–14 base-pair DNA complexes with
PME treatment of electrostatic interactions. These
MD simulations have provided us a good deal of
information on the sequence specific interaction
between ETS domain and the consensus DNA, as
schematically shown in Figure 10. Two conserved
arginine residues, Arg391 and Arg394, play an
important role in binding with the GGAA core
sequence. Although these arginine residues show
certain flexibility in side-chain conformation, they
make bidentate contacts with G1 and G2 to stabilize
the complex structure. The contacts between these
arginine residues and GG dinucleotides in the
core sequence are regulated by the motion of
Tyr395. In the high-affinity complex ETS –GGAA,
the hydrogen bonding between the hydroxyl
group of Tyr395 and the 4-carbonyl oxygen of T4
is prevented by the bulky 5-methyl group of T4 .
Rather, Tyr395 makes a contact with A3, allowing
helix-3 to be in the appropriate location in the
major groove of DNA. On the contrary, the
hydroxyl group of Tyr395 is hydrogen bonded
with the 4-nitrogen of C4 in the low affinity complex ETS –GGAG. This hydrogen bonding causes
motion in the helix-3 region and results in the disruption of bidentate contacts between the conserved arginine residues and the G1G2
dinucleotides. Thus Arg391, Arg394 and Tyr395 in
helix-3 of Ets-1 work cooperatively to recognize
the GGAA core sequence.
0
0
0
Methods
Modeling of initial structures
The crystal structure (PDB code: 1K79) of Ets-1 ETS
domain – 15 base-pair DNA complex5 was used for preparation of the starting structure of ETS– GGAA.
Although the crystal structure involved two complexes
in the asymmetric unit, these two structures are essentially identical. So, the first complex was considered in
our study. In the crystal structure, the 15 base-pair DNA
has a 50 -overhang structure, so the 50 -terminal nucleotide
in each strand was deleted. An unusual hydrogen bond
pattern was observed at base-pair 14 in the crystal structure. The glycosidic bond angle (x) at A14 was adjusted to
356
MD Simulations of Ets-1 ETS Domain–DNA Complexes
Figure 10. Schematic diagrams of the hydrogen bonds between helix-3 and core DNA sequence in (a) ETS– GGAA
and (b) ETS – GGAG complexes.
allow the base-pair 14 to form Watson– Crick type hydrogen bonds using the program INSIGHT II (version 97).
The initial structure of ETS – GGAG was built by replacing the AT base-pair at position þ4 in ETS– GGAA
with GC base-pair (Figure 2) using the INSIGHT II package so that all the conformations of other residues are the
same. The initial structure of GGAA (the 14 base-pair
DNA alone) was prepared by removing the ETS domain
from the initial structure of the ETS – GGAA complex.
The hydrogen atoms were added using HBUILD of
the CHARMM program.39 On the basis of protonation
sites, the imidazoles of His403 and His430 of Ets-1 are
protonated at both ND1 and NE2 positions in the ETS
domain – DNA complexes. The ETS domain has a charge
of þ6, while the 14 base-pair DNA includes a charge of
2 26. To neutralize the net charge of each ETS –DNA
complex, appropriate ions were placed near the phosphate oxygen atoms of the DNA and also near the solvent-exposed charged residues of Ets-1. For the
simulation on GGAA, 26 Naþ were added 3.5 Å away
from the phosphorous atom in each strand of the duplex.
Then, each system was minimized for 50 steps with steepest descent (SD) method.
Molecular simulations
MD Simulations were performed using the program
CHARMM (version c27b4)40 with all-atom force field
parameters.41 Periodic boundary conditions were defined
using
an
orthorhombic
box
of
dimensions
67.4 Å £ 62.7 Å £ 54.3 Å for ETS– GGAA and ETS –
GGAG structures, and 45.8 Å £ 63.8 Å £ 44.8 Å for
GGAA filled with TIP3P42 model water molecules. The
water molecules in the box were minimized for 100
steps of SD method and equilibrated for a period of
30 ps constant pressure– temperature (NPT) dynamics.
Then the water box was overlaid onto the Ets-1 ETS
MD Simulations of Ets-1 ETS Domain–DNA Complexes
357
domain – DNA complex with ions and crystal waters.
Solvent molecules with oxygen atoms within 1.6 Å of
non-hydrogen atoms in the DNA helices and those
within 2.5 Å of any other non-hydrogen atoms were
deleted. The total number of atoms was 24,959 for ETS–
GGAA, 24,961 for ETS – GGAG and 14,479 for GGAA.
Positions of water molecules were minimized for 100
steps of SD followed by 400 steps of adopted basis Newton –Raphson (ABNR) methods in each structure, keeping all solute molecules fixed. After that, the constraints
on ions were released, and the minimization for 100
steps of SD followed by 400 steps of ABNR methods
were performed. Then, the entire system was minimized
for 100 steps with SD and 2000 steps with ABNR
methods before starting simulations.
Leapfrog Verlet integration scheme43 was used with an
integration time-step of 1.5 fs. SHAKE44 was applied to
all covalent bonds involving hydrogen atoms. Images
were generated using the CRYSTAL module of
CHARMM. A constant dielectric of unity was used. Electrostatic interactions were treated with PME
formalism45,46 as implemented47 in the CHARMM program. PME calculations were performed using real
space cutoff of 10 Å with Lennard– Jones interactions
truncated at the same distance. A convergence parameter
(k) of 0.36 Å21 and a sixth degree B-spline interpolation
were used with the PME method.
During the equilibration, the structure was relaxed in
stages, so that the most strained parts of the system
could adjust without artifacts. Initially, harmonic constraint of 100 kcal mol2 Å22 was applied to atoms other
than waters and 21 ps simulation was performed at
298 K. Then, the constraints on ions were released and
the system was heated gradually from 0 K to 298 K, at
increments of 100 K, each for 21 ps. Next, all the constraints on solute molecules were removed and NOE
(nuclear overhauser effect)-like distance constraints
were applied on the Watson – Crick hydrogen bonds at
the 30 and 50 end base-pairs of the DNA to reduce the
end effects of DNA. Then, the system was re-equilibrated
by heating the entire model at increments of 50 K for
21 ps each from 0 K to 298 K. These stages were carried
out in NPT ensemble (with temperature and pressure of
298 K and 1 atm, respectively), so that the water box
could equilibrate in accord with the number of water
molecules. The dimensions of water box were allowed
to vary in all directions. For subsequent simulations, the
constant volume – temperature ensemble was used, as it
provides more stable trajectories.48 Then, an additional
30 ps of simulation was run at 298 K, to equilibrate the
entire system at this temperature. The heating and equilibration phases of dynamics lasted a total period of 240 ps
for each system. The production simulation was then
continued, at an average temperature of 298 K. The
simulations for ETS – GGAA and ETS– GGAG were performed for total durations of 3.5 ns and 3.9 ns, respectively, while the simulation for GGAA helix was carried
out for 3.3 ns.
complex, and 900– 3930 ps in ETS– GGAG. The experimental positional fluctuations were obtained using the
equation ðDr2 Þ1=2 ¼ ð3B=8p2 Þ1=2 from Debye – Waller
B-factors of Ets-1 ETS domain – DNA complex solved at
2.4 Å resolution (PDB code: 1K79).5 The averaged structure was obtained by least squares fitting of all the
atoms of the complex saved at 0.75 ps interval from the
trajectories to the minimized structure. Such averaged
structures were minimized for 500 steps of SD for molecular plots, drawn using the programs MOLSCRIPT,49
Raster3D50 and MIDAS PLUS.51,52
DNA bending angle u, defined as the angle between
normal vector of base-pair 2 2 and that of base-pair
614,28 (Figure 5(a)) was evaluated using the program
Freehelix98.31,32 According to the literature,14 the major
and minor groove widths were defined as the distances
between phosphorous atoms in different strands separated by 3 – 4 base-pairs, i.e. P0 ði 2 2Þ· · ·Pði þ 2Þ across the
minor groove with three intervening base-pairs and
Pði 2 2Þ· · ·P0 ði þ 2Þ across the major groove with four
intervening base-pairs. Here, PðiÞ and P0 ðiÞ are the 50 phosphates of the complementary nucleotides that comprise base-pair i; with the prime used to denote the
complementary strand.
Structural analysis
The RMSD values of the Ets-1 ETS domain were evaluated by least square fitting the backbone heavy-atoms
to the minimized structure, while for DNA helices all
the heavy-atoms excluding the end base-pairs were considered. The positional fluctuations of Ca in the ETS
domain backbone were calculated from MD trajectory,
averaged over the period 900– 3480 ps in ETS – GGAA
Acknowledgements
We thank Sun Hur (UCSB) for helpful discussions. This work was supported by NIH grant
5R37DK0917136. We acknowledge computer time
on UCSB’s SGI Origin 2000.
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Edited by B. Honig
(Received 14 February 2003; received in revised form
5 May 2003; accepted 3 June 2003)
Supplementary Material for this paper comprising one Table and one Figure is available on
Science Direct