Importance of local interactions for the stability of inhibitory helix 1 in apo Ets-1
Aleksandra Karolak, Arjan van der Vaart
Department of Chemistry, University of South Florida, 4202 East Fowler Avenue CHE 205, Tampa, FL 33620, United States
► Simulation of Ets-1 constructs
identiﬁed how HI-1 is stabilized
in the apo state.
► Contacts between HI-1 and HI-2/H4
are important for the stability of HI-1.
► Removal of a few local contacts may
lead to the partial unfolding of Ets-1.
Received 3 February 2012
Received in revised form 10 March 2012
Accepted 18 March 2012
Available online 23 March 2012
Molecular dynamics simulation
Inhibitory helix 1 (HI-1) of the Ets-1 human transcription factor unfolds upon binding the target DNA sequence.
To identify the interactions that stabilize HI-1 in the apo state, we performed replica exchange and molecular
dynamics simulations of various apo Ets-1 constructs. The simulations indicate the importance of local interac-
tions for the stability of HI-1. The HI-2 and H4 helices stabilize the helical state of HI-1 through speciﬁcresi-
due–residue contacts and macrodipolar interactions. The amount of stabilization in small length HI-1+H2
and HI-1+H4 constructs was similar to that in the protein. The studies suggest that the partial unfolding of
Ets-1 upon DNA binding can be achieved by the removal of just a few speciﬁc local contacts.
© 2012 Elsevier B.V. All rights reserved.
The human Ets-1 transcription factor is important for embryonic
development , apoptosis , and angiogenesis  in normal and path-
ological growth. Ets-1 is also involved in cancer metastasis and tumor pro-
gression.Highexpressionlevelsinbreast[4,5],ovary[6–8], and cervix 
tumors correlate strongly with bad prognosis, while elevated expression
is relevant for lung ,colon,pancreatic[12,13], thyroid [13,14],
and oral  cancers. In addition, Ets-1 plays a role in immunity and
autoimmune diseases [16,17]. The protein consists of six domains .
The N-terminal domain contains a RAS-responsive phosphorylation site
[19,20], which regulates the transcriptional activity of Ets-1. This
domain is followed by the pointed domain, important for protein–pro-
tein interactions , the transactivation domain, important for
transcription activation , and the D, ETS, and F domains which reg-
ulate DNA-binding [18,23].
DNA is bound by a winged helix-turn-helix motif in the ETS-domain
(residues 331–415) . This highly conserved domain binds the
GGAA/T sequence in the major groove of purine-rich DNA by insertion
of the recognition helix (H3). The minor groove is bound by a loop
between β-sheets S3 and S4 and the turn between α-helices H2 and
H3. The binding afﬁnity for DNA is modulated by an auto-inhibitory
module, which ﬂanks the ETS domain and decreases the binding afﬁnity
of DNA 10 to 20 fold compared to the bare ETS domain .The
auto-inhibitory module consists of residues 301–330 of the D domain
and residues 415–440 of the F domain [25–27]. These residues are
folded into four α-helices: inhibitory helix 1 and 2 of the D domain
(HI-1 and HI-2, respectively), and H4 and H5 of the F domain.
The DNA-binding afﬁnity is further regulated by calcium-dependent
phosphorylation of an unstructured serine-rich region of the D domain
(residues 243–300) , and by binding of protein partners like
Runt-related transcription factor .
Biophysical Chemistry 165–166 (2012) 74–78
Abbreviations: MD, molecular dynamics; REX, replica exchange simulations.
⁎ Corresponding author. Tel.: +1 813 974 8762; fax: +1 813 974 3203.
E-mail address: email@example.com (A. van der Vaart).
0301-4622/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
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