Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA

Abstract

The [2Fe-2S] transcription factor SoxR, a member of the MerR family, functions as a bacterial sensor of oxidative stress such as superoxide and nitric oxide. SoxR is activated by reversible one-electron oxidation of the [2Fe-2S] cluster and then enhances the production of various antioxidant proteins through the soxRS regulon. In the active state, SoxR and other MerR family proteins activate transcription from unique promoters, which have a long 19- or 20-bp spacer between the −35 and −10 operator elements, by untwisting the promoter DNA. Here, we show the crystal structures of SoxR and its complex with the target promoter in the oxidized (active) state. The structures reveal that the [2Fe-2S] cluster of SoxR is completely solvent-exposed and surrounded by an asymmetric environment stabilized by interaction with the other subunit. The asymmetrically charged environment of the [2Fe-2S] cluster probably causes redox-dependent conformational changes of SoxR and the target promoter. Compared with the promoter structures with the 19-bp spacer previously studied, the DNA structure is more sharply bent, by ≈1 bp, with the two central base pairs holding Watson–Crick base pairs. Comparison of the target promoter sequences of the MerR family indicates that the present DNA structure represents the activated conformation of the target promoter with a 20-bp spacer in the MerR family.

Reactive oxygen species (ROS) provide cellular and genetic damages to aerobic organisms, and cells have thus evolved defense systems against oxidative stress (1–3). In Escherichia coli, the soxRS regulon mediates the response to superoxide and nitric oxide by activating various defense genes (4–6). The soxRS regulon is induced in two steps (7, 8): first, SoxR activates the transcription of the soxS gene in response to superoxide, nitric oxide, and redox-cycling agents, and, second, the increased level of the SoxS protein, a member of the AraC family, enhances the production of various antioxidant proteins and repair proteins (9).

SoxR belongs to the MerR family of transcriptional activators (10, 11). It forms a homodimer in solution, with each 17-kDa subunit containing a [2Fe-2S] cluster (12, 13). The [2Fe-2S] cluster is essential for the activity of SoxR (14). In the absence of oxidative stress, SoxR with the reduced [2Fe-2S] cluster is inactive for transcription. Upon oxidative stress, the metal center is oxidized, and SoxR enhances the transcription of the soxS gene up to 100-fold (15–17). Nitric oxide also activates SoxR by direct nitrosylation of the [2Fe-2S] cluster (18). Apo-SoxR and reduced SoxR can bind to DNA with an affinity similar to that of oxidized SoxR, but only oxidized SoxR is able to activate the transcription of the soxS gene (14, 15). Therefore, SoxR senses oxidative stress using the redox states of the [2Fe-2S] cluster and regulates the transcription of the soxS gene by structural changes between the oxidized and reduced forms. In vivo, the oxidized SoxR is reduced rapidly and maintained in its reduced state by specific enzymes (19, 20).

SoxR binds to a dyad-symmetric sequence between the −35 and −10 elements of the soxS promoter. Both elements of the soxS promoter are separated by an unusual 19-bp spacer, compared with the optimal 17-bp spacer in σ⁷⁰-RNA polymerase (RNAP) targeted promoters (14). The target promoters of the MerR family also have a long 19- or 20-bp spacer (21), which prevent open complex formation by RNAP without an activator. The MerR family includes metal-stress sensor proteins such as MerR (22), ZntR (23, 24), and CueR (24, 25), and activator proteins of multidrug transporters, such as BmrR (26, 27) and Mta (28, 29). Furthermore, with recent progress in genome sequence data, many MerR family members have been identified in bacteria genomes (21). These members of the MerR family have a sequence similarity in an N-terminal DNA-binding domain and form a homodimer by antiparallel coiled coil (24, 27, 29). In response to specific signals, MerR family members are assumed to distort the conformation of their target promoters to initiate transcription by RNAP (22, 23, 30). Structural evidence of the DNA distortion mechanism for transcriptional activation is provided by the crystal structures of BmrR and MtaN bound to their target promoters (27, 29). These structures reveal that the promoters with the 19-bp spacer are bent sharply at the center with base pair breaking and sliding, resulting in the remodeling of the −35 and −10 elements on the same face of the DNA helix (27, 29). However, it is not yet clear how promoters with a 20-bp spacer is distorted for transcriptional activation.

To obtain further insights into the redox regulation mechanism by SoxR, we have determined the crystal structures of SoxR from E. coli and its complex with the soxS promoter in the oxidized state at 3.2-Å and 2.8-Å resolution, respectively [supporting information (SI) Table 1]. These results provide deep insights into the redox-dependent gene regulation by an iron-sulfur cluster and structural evidence of the general mechanism of transcriptional activation by the MerR family.

Results

Overall Structure of the SoxR–soxS Promoter Complex.

The overall structure of SoxR consists of a DNA-binding domain (residues 1- 80), a dimerization helix (α5) (residues 81–118), and an Fe-S cluster-binding domain (residues 119–154) (Fig. 1 A and B). The dimerization α5-helix (and α5′-helix, where the prime indicates the other subunit) forms an antiparallel coiled coil stabilizing the SoxR dimer. The overall architecture of the SoxR–DNA complex is similar to those of other MerR family proteins (24, 27, 29). However, the arrangement of the α5-helix and the DNA-binding domain is quite distinct from the others (Fig. 1C and SI Fig. 5). Compared with BmrR, the α5-helix is ≈20° rotated and ≈7 Å translated. Consequently, the DNA-binding domain of the other subunit of SoxR is located at a higher position than that of BmrR. This unique position of the α5-helix of SoxR is stabilized by hydrophobic interactions between conserved residues of the α5-, α3-, and α4-helices (Fig. 1D and SI Fig. 6). Trp-98 is stabilized by van der Waals contacts with Ile-59, Ala-63, and Ile-68. Additional van der Waals interactions occur between Leu-94, Ala-76, and Phe-77; and between Leu-86, Tyr-56, and Ile-59 (Fig. 1D). Conservation of these residues suggests that the specific position of the α5-helix is conserved among SoxR proteins.

Fig. 1.

Overall structure of the SoxR–soxS promoter complex. (A) Stereoview of the overall structure of the SoxR–DNA complex. The SoxR dimer is shown in a ribbon representation and the DNA fragment appears in a stick model. The DNA-binding domain, dimerization helix, and Fe-S cluster binding domain are shown in blue, magenta, and yellow, respectively. The two iron and two sulfur atoms of the [2Fe-2S] cluster are represented with brown and green spheres, respectively. The palindromic sequence of the 20-bp DNA fragment is shown in Lower, where the soxS promoter sequences are highlighted in blue. (B) Ribbon representation of the SoxR monomer. (C) Cα backbone diagram of a superposition of the overall structures of SoxR (blue) and BmrR (red). The DNA-binding domain (DBD) of one subunit of SoxR is superimposed on that of BmrR. To clarify, the drug-binding domain of BmrR is omitted. (D) Conserved interactions between the α5-helix and DNA-binding domain of SoxR.

The crystal structure of the DNA-free form of SoxR also was determined at 3.2-Å resolution. Upon binding to DNA, the DNA-binding domain undergoes an outward rotation of approximate 9°, and the Fe-S cluster-binding domain receives an outward rotation of ≈6°, resulting in a widening of the distance between the α2- and α2′-helices from 29.3 to 31.5 Å (SI Fig. 7). The α5-helix connecting both domains shows an inner helical twist, which leads to a change in the relative positions of the dimerization helices. These observed changes of SoxR are different from those observed in MtaN (29). In the MtaN–mta promoter complex, only a hinge movement is observed. However, the observed movement of MtaN may result from the missing of a sensor domain of MtaN, which probably interact with the DNA-binding domain of the other subunit. Therefore, similar conformational changes as observed in SoxR are expected to occur in other MerR family proteins during binding to their target promoters.

Asymmetric Environment of the [2Fe-2S] Cluster of SoxR.

The [2Fe-2S] cluster of SoxR is coordinated by conserved four cysteine residues (Cys-119, Cys-122, Cys-124, and Cys-130) (Fig. 2A). This CX₂CXCX₅C motif for the binding of the [2Fe-2S] cluster is different from those formed in the other [2Fe-2S] proteins. The remarkable feature of the [2Fe-2S] cluster of SoxR is its asymmetric environment (Fig. 2 A and B). The lower sulfur atom (S1) of the [2Fe-2S] cluster is hydrogen-bonded to the amide of Gly-123 and makes van der Waals contacts with the amides of Cys-124 and Leu-125, with the O atom of Cys-119 and with the Cα atoms of Cys-119 and Cys-122. However, the upper sulfur atom (S2) makes van der Waals contacts with the O atom of Asp-129 and with the C atoms of Cys-130 and Pro-131. Although the side chain of Ser-126 is disordered, it might be involved in the interaction with the S2 atom. Compared with other [2Fe-2S] proteins, and despite the limited resolution (2.8 Å), the plane of the [2Fe-2S] cluster is shown to be not vertical but to bend by ≈20° to that of the four sulfur atoms of the cysteine residues (Fig. 2A). The S2 atom and two Fe atoms are fully exposed to the solvent (Fig. 2B). The solvent-exposed environment and intermolecular interactions (see below) probably determine the redox potential of SoxR (−285 mV) (15, 16, 31). In vivo, SoxR is actively maintained in the reduced state by specific enzymes (19, 20). The solvent-exposed [2Fe-2S] cluster likely enables fast electron transfer between SoxR and various redox agents, suggesting that SoxR responds not only to superoxide directly but also to multiple signals that indicate possible oxidative stress (32, 33). The solvent-exposed environment also allows the direct nitrosylation of the [2Fe-2S] cluster and the rapid dispose of this modification (18).

Fig. 2.

Asymmetric environment of the [2Fe-2S] cluster of SoxR. (A) Stereoview of the [2Fe-2S] cluster environment in stick representation. Iron and sulfur atoms are indicated by brown and green spheres, respectively. The electron density of a F_o−F_c omit map calculated by omitting the two sulfur atoms of the [2Fe-2S] cluster is shown at 6σ (red). NH-S hydrogen bonds are represented in orange broken lines. (B) Surface representation of the Fe-S cluster-binding domain. Iron and sulfur atoms of the [2Fe-2S] cluster and cysteine residues are colored in brown and green, respectively. (C) Stereoview of the interactions between the Fe-S cluster-binding domain and the DNA-binding domain of the other subunit. The other subunit is shown in white.

The Fe-S cluster-binding domain is further stabilized by interaction with the α3′-and α5′-helices of the other subunit (Fig. 2C). Leu-112, Leu-116, Leu-125, and Leu-132 undergo hydrophobic interactions with Ile-59′, Ile-62′, Trp-91′, and Ser-95′. The Nε1 atom of Trp-91′ also forms a hydrogen bond with the O atom of Cys-119. In addition, the NH1 and NH2 of Arg-55′ form hydrogen bonds with the O atoms of Gly-123 and Cys-124. Arg-65′ also forms hydrogen bonds with the O atoms of Leu-132 and Oγ1 of Glu-115. These residues are highly conserved among SoxR proteins (SI Fig. 6).

Distorted DNA Structure.

The overall DNA structure of the SoxR–DNA complex is a bent conformation with local untwisting (Figs. 1A and 3A). Although the half-site of the structure of the SoxR–DNA complex is similar to those of BmrR and MtaN, the DNA in the SoxR–DNA complex is further bent ≈65° at the middle away from the protein, compare to ≈47–50° in BmrR and MtaN (Fig. 3 A and B). In the crystal structures of BmrR and MtaN in complex with their promoters, the central base pairs of the bmr and mta promoters break and slide away from each other (27, 29). However, the two central Ade1-Thy1′ and Thy1′-Ade1 base pairs of the soxS promoter hold Watson–Crick base pairs (Figs. 1A and 3C). The rise, roll, and twist values of the central base step between A1/T1′ and T1′/A1 of the soxS promoter are 3.1 Å, 12.5°, and 43.9°, respectively. In addition, bases 6 to 10 are bent ≈15° toward the α2-helix. As a result, the overall end-to-end length of the 20-bp DNA fragment is further shortened by ≈3.4 Å, which corresponds to 1 bp, compared with those of the activated bmr and mta promoters (Fig. 3B). This significant difference between SoxR and BmrR/MtaN is attributable to the promoter sequence. The bmr and mta promoters have one central-base pair separation between the pseudopalindromic sequences (27, 29). In contrast, the soxS promoter has a complete palindromic sequence in which the two central-base pairs are inserted between two regions that interact with SoxR.

Fig. 3.

Activated conformation of target promoter of SoxR. (A) Side and top views of the overall structure of the soxS promoter with the global DNA helical axis (cyan line) (46). (B) Comparison of the 20-bp promoter structures of SoxR (blue) and MtaN (red). Two promoter structures are superimposed on each half-site of DNA. (C) The electron density of simulated annealing omit map (20- to 2.8-Å resolution) around the middle of the promoter is shown at 1.5σ. Thy1′ and Ade1 are indicated.

Interaction with DNA and Promoter Recognition.

Protein–DNA interactions are formed primarily by hydrogen bonds and van der Waals contacts between residues of the wing helix–turn–helix motif and the DNA backbone of Ade2 to Thy5 and Thy7′ to Cyt9′ (SI Fig. 8 A and B). These interactions are similar to those observed in the BmrR or MtaN–DNA complex. Base-direct contacts occur at only three residues (SI Fig. 8 B and C). Phe-30 makes van der Waals contacts with Cyt3 (3.6 Å between the Cε1 of Phe-30 and C5 of Cyt3). The phenyl ring of Phe-30 is perpendicular to the pyrimidine ring of Cyt3. This interaction presumably discriminates between cytosine and thymine. An additional base-direct interaction is observed at the van der Waals contact between His-29 and Thy7. In the MerR family proteins, residues at positions 29 and 30 are His/Arg and Phe/Tyr, respectively (SI Fig. 6), and similar base-direct interactions are observed in the DNA-bound structure of BmrR and MtaN (SI Fig. 9) (29). Therefore, base-direct interactions by these two conserved residues are presumably a common feature among the MerR family to stabilize the distorted DNA conformation. Cβ of Ser-26, which is completely conserved among SoxR proteins, makes van der Waals contacts with the methyl groups of Thy4 and Thy5. Specifically, each MerR family protein has an intrinsic residue at position 26 (serine for SoxR, glutamate for MerR, lysine for CueR, etc.) (SI Fig. 6). Similar base-specific interactions also are observed in the crystal structures of the DNA complexes of BmrR and MtaN (SI Fig. 9) (29). These observations suggest that the MerR family members recognize their promoter sequence using the intrinsic residue at position 26.

Discussion

The overall structure of SoxR is shown to be similar to those of other MerR family proteins, but the domain arrangement is distinct (Fig. 1). The specific position of the dimerization helix of SoxR is stabilized by hydrophobic interactions between conserved residues. These results imply that the specific sensor systems of each MerR protein are sophisticatedly constructed by the arrangement and length of the dimerization helix and by various sensor domains. The structures reveal that the Fe-S cluster-binding domain directly interacts with the DNA-binding domain through interactions between highly conserved residues (Fig. 3C), suggesting that signal for oxidative stress is transmitted from the [2Fe-2S] cluster to the DNA-binding domain. Completely conserved Arg-55 and Trp-91 interacting cysteine residues are presumably important for the redox signaling of SoxR. Similar interactions between the metal-binding domain and the DNA-binding domain of the other subunit also are observed in CueR and ZntR (24), supporting the idea that the signal transduction in the MerR family is transmitted through direct interaction between the sensor domain and the DNA-binding domain.

The structure of the SoxR–DNA complex provides a structural rationalization for the previous mutation analysis (34, 35). Mutant proteins Y31H, L36V, I62V, and I73F are DNA-binding defective proteins, and the [2Fe-2S] cluster in these mutant proteins is unstable. I62N will disrupt hydrophobic interactions between the DNA-binding domain and the Fe-S binding domain (Fig. 2C). Other mutations will lead to disruption of the hydrophobic core of the DNA-binding domain and consequently affect interactions between two domains. As a result, these mutations will weaken the stability of the [2Fe-2S] cluster. Ile-106 and Leu-109 are located at the middle of the coiled coil, forming a hydrophobic patch between each subunit. A mutation that was defective in DNA binding and transcriptional activation, I106T (34), will disrupt these hydrophobic interactions and affect the conformation of the coiled coil. Two other activation-defective mutants are L94P and S95P (34), which are located in the dimerization helix α5. The side chain atoms of Leu-94 are involved in the interaction between the dimerization helix α5 and helices α4 and α3 of the DNA-binding domain. Ser-95 interacts with the α5′-helix from the other subunit. Replacing these residues with proline will change the relative positions of the α5-helix, the DNA-binding domain, and the Fe-S binding domain from other subunit. Therefore, the proper arrangement of the DNA-binding domain of each subunit is essential for redox signal transduction. Other intriguing altered redox mutant is R20C (31). Arg-20 is completely conserved among SoxR proteins and is located at the accessible molecular surface. The position of Arg-20 and a constitutively active property in vivo of R20C suggests that Arg-20 is involved in interactions with a SoxR-specific reductase such as RsxC (20).

Redox-Dependent Structural Regulation of SoxR.

An interesting question about the redox sensing of SoxR is how the redox change of the [2Fe-2S] cluster of SoxR causes a large conformational change of SoxR and the target promoter. The answer is probably provided by the asymmetric charge distribution of the [2Fe-2S] cluster environment. In ferredoxins and putidaredoxins, the [2Fe-2S] cluster is shielded from the solvent by surrounding residues (36, 37). Both sulfur atoms of the [2Fe-2S] cluster receive similar hydrogen bonds from the main chain amide. By contrast, the [2Fe-2S] cluster of SoxR is located on the molecular surface. The one side of the cluster is surrounded by the positively charged three amides of the main chain, whereas the other side is unable to interact with any amide but with the negatively charged carbonyl oxygen atom (Fig. 2A). Upon reduction, an additional negative charge on the sulfur atoms will attract the amides of the main chain and increase the charge repulsion with the oxygen atom, possibly resulting in large conformational changes (SI Fig. 10). This asymmetric conformational changes will result in an outward movement of the C-terminal region of SoxR and subsequently change the relative position of the DNA-binding domains through the direct interactions between the DNA-binding and [2Fe-2S] cluster binding domains. As a result, the reduced form of SoxR will provide a further twist in the DNA and separates both promoter elements from the same face.

Activated Conformation of the Target Promoter with a 20-bp Spacer of the MerR Family.

This study reveals that the DNA structure is distorted and unwound by ≈3-bp, compared with B-form DNA (Fig. 3). The DNA structure of SoxR is distinct from those previously reported in the BmrR–DNA and Mta–DNA complexes, because of difference in their promoter sequence. Based on these observations, the target promoters of the MerR family can be categorized into two groups: 1-bp separation and 2-bp separation groups (SI Fig. 11). Promoters of the 1-bp separation group have a 1-bp separation between dyad-symmetric sequences and a 19-bp spacer between the −35 and −10 elements. The 2-bp separation group includes promoters that have a 2-bp insertion between dyad-symmetric regions. Although most promoters of the 2-bp separation group have a 20-bp spacer, the soxS and mer promoters, which have a 19-bp spacer, also belong to the 2-bp separation group. These observations indicate that the present DNA structure of the SoxR–DNA complex exhibits the active conformation of the target promoters with a 20-bp spacer. This suggestion is consistent with previous DNA footprint studies on MerR, SoxR, and ZntR (14, 22, 23). The DNA footprint results showed that similar positions of the symmetrical promoter regions (base 7 or 8) are hypersensitive to DNase I, indicating a similar conformation is formed in their target promoters.

The structure of the soxS promoter shows a target promoter with a 20-bp spacer is distorted for transcriptional activation in the MerR family. In a 20-bp spacer on B-form DNA, the −35 and −10 elements are separated by 10.2 Å and rotated by 108° compared with those in the optimal 17-bp spacer (Fig. 4), and therefore RNAP cannot bind both elements. In contrast, the DNA distortion observed in the SoxR–DNA complex results in the remodeling of the −35 and −10 elements positions similar to those in the 17-bp spacer, allowing RNAP to initiate transcription (Fig. 4). For SoxR, the relative positions of both elements in the active state correspond to those in promoters with a 16-bp spacer, which can be substrates for RNAP. On the mutant spacer of the soxS promoter, the shortened promoter structure will prevent the binding of RNAP to the operator elements (30). RNAP will bind the promoter on the opposite site of the binding site of SoxR, indicating that SoxR may not interact directly with RNAP. The MerR family has attracted considerable attention because of their high selectivity and sensitivity for heavy metal ions (38). Our results will contribute to development of MerR family proteins-based biosensors of heavy metal, oxidative stress, and organic compounds.

Fig. 4.

Structural framework of transcriptional activation by DNA distortion. Comparison of the relative positions of the −35 and −10 elements on B-form DNA with 20-bp (A) and 17-bp (B) spacers and those on the distorted DNA with 20-bp (C) and 19-bp (D) spacers. The distorted DNA model (C and D) is built based on the present structure of the SoxR–soxS promoter.

Materials and Methods

Crystallization and Data Collection.

The purification and crystallization of SoxR and the SoxR–DNA complex were performed as described in ref. 39. The x-ray diffraction data of the SoxR–DNA complex were collected at the BL41XU and BL44B2 beamlines at SPring-8 and at the NW12 beamline at Photon Factory-Advanced Ring (PF-AR) and were processed with the HKL package (40). Spectral data were measured at 100 K with a microspectrophotometer at the BL44B2 beamline.

Structure Determination and Refinement.

The structure was determined by the single-wavelength anomalous dispersion (SAD) method using a dataset collected at a wavelength of 1.6000 Å. The positions of each Fe atom of the [2Fe-2S] cluster were fitted into the anomalous difference Fourier map. The initial phases were calculated by using SOLVE (41) and further improved by RESOLVE (42). Manual model building was performed by using O (43). Simulated annealing, energy minimization, and B factor refinement were carried out by CNS (44). Cycles of the manual modeling and structure refinement of CNS were performed. To obtain the structure of the fully oxidized form, eight datasets in which only eight frames per set (≈80 s) were collected from one crystal (nearly 100% oxidized states) were merged and processed, because the [2Fe-2S] cluster of SoxR was rapidly reduced during data collection during exposure to synchrotron radiation (data not shown). The structure of SoxR was determined by molecular replacement using MOLREP (45). The structure of the SoxR–DNA complex was used as a search model. The final model (R_work of 24.3%, R_free of 28.1%) of the oxidized SoxR–DNA complex comprises residues 10–79 and 85–136, one [2Fe-2S] cluster, 22 water molecules, one glycerol molecule, and half a molecule of DTT. Ramachandran plots of the final model of the SoxR–DNA complex shows 90.4% of the residues in the most favored regions, and 9.6% in the allowed regions. All structural figures were made with PyMOL (http://pymol.sourceforge.net/).