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The oxidative-stress sensor SoxR bound to DNA

Gian Garduque '12 and Jill Pattison '12


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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