The
oxidative-stress sensor SoxR bound to DNA
Gian Garduque '12 and Jill Pattison '12
Contents:
I.
Introduction
SoxR is a
154 amino acid DNA binding protein which serves as an oxidative stress
sensor. SoxR
and SoxS
are responsible for induction of the soxRS
regulon. In response to
superoxide, nitric oxide, and redox-cycling agents, SoxR activates SoxS
transcription. Increased levels of SoxS enhance the production of
antioxidant
and repair proteins. SoxR regulates SoxS transcription by structural
changes
occurring between oxidized and reduced forms. SoxR binds between the
-35 and
-10 elements of the soxS
promoter, which are separated by a 19 bp
spacer. Due to this unusually large spacing, SoxR distorts the
conformation of
the soxS
to initiate RNAP transcription, similar to other members of the
MerR family. This tutorial presents the structure of SoxR from E.
coli
bound to DNA and the [2Fe-2S] complex.
II.
General Structure
The overall
structure of SoxR
is similar to structures of other MerR family proteins. SoxR functions
as a
dimer
.
The
dimerization helix
(α5)
forms an antiparallel
coiled coil (α5 from one monomer
and α5'
from
the other
monomer),
which
serves to stabilize the dimer
.
In addition to the dimerization helix, two distinct domains make up
the SoxR monomer:
the DNA
binding
domain
and
the Fe-S
cluster-binding domain
.
The DNA binding
domain of
SoxR is located at a higher position than other MerR proteins, due to
rotation
of the α5-helix. This
position is stabilized by hydrophobic
interactions between the α5
, α3
, and
α4
helices
.
Upon binding DNA,
the DNA-binding domain
rotates
outward 9°, and the Fe-S cluster binding domain rotates outward
6°. These
rotations result in a greater distance between the α2
and α2'
helices (29.3
to 31.5 Å)
.
III.
DNA Binding Domain
Associations
between SoxR and DNA
consist primarily of hydrogen bonds and van der
Waals interactions between the wing
helix-turn-helix motif and
the DNA
backbone
. Three
amino
acid residues of each SoxR monomer contact
the DNA bases directly
.
These
include: Phe-30
contacting Cyt3
and Ade2
,
His-29
contacting Thy7’
,
and Ser-26
contacting Thy4
and Thy5
.
The phenyl
ring of Phe-30
is
perependicular to the
pyrimidine ring of Cyt3
.
Van der Waals contacts
between Phe-30
and Cyt3
allow Phe-30
to
discriminate between cytosine and thymine
.
The yellow C
atom in Cyt3
is where
a methyl group would branch out if this base were a thymine. His-29
interacts with Thy7’
through van der Waals
forces
.
Ser-26
contacts the methyl
groups of Thy4
and Thy5
through van
der Waals interactions
.
Because other
proteins in the MerR
family also use the amino acid residue at position 26, Ser-26 in
SoxR
,
to recognize DNA
in this base-specific fashion, it is believed that the amino acid at
this position is used by MerR family proteins to recognize specific
promoter sequences.
Each
monomer of SoxR also makes multiple contacts to the DNA backbone in two
clusters, one of which is closer to the center of the dimer and the
other which is near the outside of the dimer. At the inner
region, Ser-26,
Ala-27,
Tyr-31,
Gln-64,
and Leu-70
all
make hydrogen
bonds to phosphate groups of
the DNA backbone (O
atoms of phosphate groups are
hydrogen bond acceptors in each case), Ala-24
and Pro-69
have
van der Waals interactions with the
phosphates of the backbone,
and Leu-70
maintains
van der Waals contacts with the ribose of Ade2
.
At the outer region, Arg-47,
His-29,
and Gly-15
hydrogen
bond
with phosphate groups in the DNA
backbone (O
atoms of phosphate groups are
H bond acceptors),
Arg-41,
Gln-46,
Pro-14,
and Thr-13
make
van der Waals
contacts with phosphates
of the backbone,
and Arg-41,
Asn-45
,
and Gln-46
maintain
van der Waals interactions with
ribose sugars
of the backbone
.
IV.
DNA Distortions
As
a result of the SoxR dimer
binding
to
DNA, the DNA is bent ~65°
at the center away from the protein
.
Despite this
bend, the
central A-T and T-A base pairs, Ade1-Thy1’
and
Thy1’-Ade1,
maintain Watson-Crick base pairing
(Hydrogen
bond
donor, hydrogen bond
acceptor,
hydrogen bond pairs connected with a line).
Bases
6 through 10 are also bent
~15° inward toward the α2
helix
.
The
combined effect of these distortions is the scrunching of the 19-bp
spacer between the -10 and -35 elements of the soxS
promoter to a length that mimics that of an
ideal 17-bp spacer
.
V.
Fe-S Cluster-Binding Domain
The
Fe-S
cluster bound to each of the monomers consists of two Fe atoms
and two
S atoms
.
The cluster,
located at the C terminus of the
protein, is coordinated by four cysteine residues, Cys-119,
Cys-122,
Cys-124,
and Cys-130
.
The lower sulfur atom (S1)
forms a hydrogen bond
with the amide of Gly-123
and has van der Waals interactions with the
amides of Cys-124
and Leu-125,
with the oxygen of Cys-119,
and with the
C atoms of Cys-119
and Cys-122
.
The upper
sulfur atom (S2)
has van der Waals interactions with the backbone oxygen of Asp-129
and
the C atoms of Cys-130
and Pro-131
.
All four atoms of the Fe-S
cluster, to some degree, are exposed to the solvent
.
This
exposure allows for rapid electron
transfer
between SoxR and various
redox agents, which allows SoxR to respond quickly to the presence of
these agents in the cell.
The Fe-S cluster is actively
maintained in the reduced state by cellular enzymes, and this aids in
the response of SoxR to oxidizing agents by ensuring that the Fe-S
cluster is always
in a state capable of being oxidized.
The
α3’
and α5’
helices of the
other SoxR monomer interact with and provide stabilization to the Fe-S
cluster-binding domain and to
sections of the α5
helix
adjacent to the Fe-S
cluster-binding domain
. Leu-112,
Leu-116,
Leu-125,
and Leu-132
maintain hydrophobic
interactions with Ile-59’,
Ile-62’,
Trp-91’,
and Ser-95’
.
Hydrogen
bonds include: the ring N
of Trp-91’
bonded with the oxygen
of Cys-119
(H
bond
donor, H
bond acceptor)
two
terminal
N
moieties (NH1
and NH2)
of the side chain of Arg-55’
bonded
with the oxygen atoms
of Gly-123
and Cys-124,
NH1
and NH2
of Arg-65’
bonded
to the oxygen
of Leu-132
and to one of the
side chain oxygen atoms
of Glu-115
. It
is thought
that these
interactions allow the presence
of oxidative stressors to be signaled
through conformational changes from the Fe-S cluster-binding domain to
the DNA binding domain. This signaling allows SoxR to alter
the conformation of the promoter DNA in response to the oxidative state
of the Fe-S cluster.
VI.
References
Amabile-Cuevas,
C. F. and B. Demple. 1991. Molecular
Characterization of the SoxRS Genes of Escherichia Coli: Two Genes
Control a
Superoxide Stress Regulon. Nucleic
Acids Research 19(16): 4479-484.
Brown,
N. L., J. V. Stoyanov, S. P. Kidd, and J. L. Hobman. 2003. The MerR
family of
transcriptional regulator. FEMS
Microbiology Reviews
27:
145-163.
Watanabe,
S., A. Kita, K. Kobayashi, and K. Miki. 2008. Crystal Structure of the
[2Fe-2S] Oxidative-stress Sensor SoxR Bound to DNA. Proceedings
of the National Academy of Sciences
105(11): 4121-126.
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