A conserved structural module regulates transcriptional responses to diverse stress signals in bacteria

Abstract

A transcriptional response to singlet oxygen in Rhodobacter sphaeroides is controlled by the group IV sigma factor sigma(E) and its cognate anti-sigma ChrR. Crystal structures of the sigma(E)/ChrR complex reveal a modular, two-domain architecture for ChrR. The ChrR N-terminal anti-sigma domain (ASD) binds a Zn(2+) ion, contacts sigma(E), and is sufficient to inhibit sigma(E)-dependent transcription. The ChrR C-terminal domain adopts a cupin fold, can coordinate an additional Zn(2+), and is required for the transcriptional response to singlet oxygen. Structure-based sequence analyses predict that the ASD defines a common structural fold among predicted group IV anti-sigmas. These ASDs are fused to diverse C-terminal domains that are likely involved in responding to specific environmental signals that control the activity of their cognate sigma factor.

Figure 1. Structure of σ^E/ChrR Complex

(A) Ribbon diagrams (with α helices shown as cylinders) of the σ^E/ChrR complex. Regions of each protein are color coded: σ^E₂, green; σ ^E_2–4 linker, cyan; σ^E₄, yellow; ChrR-ASD, purple; and ChrR-CLD, pink. The Zn²⁺ ions (one each in the ChrR-ASD and ChrR-CLD) are shown as green spheres, with the side chains of the protein ligands shown. An eight-residue disordered segment connecting the ChrR-ASD with the ChrR-CLD (ChrR residues 78–85) is shown as purple spheres. The α helices of the ChrR-ASD are labeled (H1–H4). (B) The conformational change of σ₄ when bound to ChrR or T4 bacteriophage AsiA. The σ₄s are shown as ribbon diagrams (σ_4.1, orange; σ_4.2, yellow). On the left is Ec σ^E₄ from the Ec σ^E/RseA complex (Campbell et al., 2003), showing the native conformation of σ₄ (Campbell et al., 2002b; Murakami et al., 2002; Vassylyev et al., 2002). In the middle is Rsp σ^E₄ from the Rsp σ^E/ChrR complex, with the ChrR-ASD shown as a transparent, purple backbone worm. On the right is Ec σ⁷⁰₄ from the Ec σ⁷⁰₄/AsiA complex (Lambert et al., 2004), with AsiA shown as a transparent, blue backbone worm. Each view is oriented so that the helical segments of σ_4.1 are aligned.

Figure 2. Zn²⁺ Coordination by the ChrR-ASD and Its Role in σ^E Inhibition

(A) ChrR-ASD coordination of Zn²⁺. The ChrR-ASD coordinates Zn²⁺ via the side chains of His6, His31, Cys35, and Cys38. The ChrR-ASD is shown as a ribbon diagram (helices H1–H3). Shown below the structure are the results of β-gal assays (in Miller units) from an rpoE::lacZ fusion in an Ec tester strain containing the indicated ChrR protein (Newman et al., 1999). Except for the data on the right, the assays were done with full-length ChrR with the indicated amino acid changes. The data on the right were obtained with cells harboring ChrR₈₅. Each data set represents the average of at least five independent assays. The bar indicates standard deviation between assays. (B) ChrR₈₅ inhibits σ^E-dependent transcription in vitro. The top panel shows the effects of adding indicated amounts of ChrR₈₅, or full-length ChrR, on σ^E-dependent transcription and the quantification of radiolabel incorporated into the rpoE P1 transcript (Newman et al., 1999). The bottom panel shows the effects of the same ChrR proteins on σ^E-independent transcription (RNA1).

Figure 3. Zn²⁺ Coordination by the ChrR-CLD

(A) ChrR-CLD coordination of Zn²⁺ via side chains of His141, His143, Glu147, and His177. The ChrR-CLD (ChrR residues 86–194) is shown as a ribbon diagram, with side chains of the Zn²⁺ ligand shown in stick format. (B) The ChrR-CLD Zn²⁺ and Cys189 are solvent exposed. A cross-section of the ChrR-CLD (cut at the level of the Zn²⁺, which is shown as a green sphere) is shown in both stick format and as a molecular surface (calculated without the Zn²⁺ by using msms with a 1.4 Å probe radius; http://www.scripps.edu/~sanner/html/msms_home.html) and color coded as follows: carbon atoms, pink; nitrogen, blue; oxygen, red; and sulfur, yellow). The thiol of Cys189 (labeled) is exposed at the bottom of the pocket. The Zn²⁺ ligand His177 is also labeled.

Figure 4. Contacts between the ChrR-ASD and CLD

(A) Residues of the ChrR-ASD that contact the ChrR-CLD (<4 Å) are shown. These are Ile3–Ser8, and Glu22–Leu34. The ChrR-ASD Zn²⁺ is shown as a green sphere. The α carbon backbone of the ChrR-ASD is shown as a purple worm, with the side chains in stick format (carbon atoms, purple; nitrogen, blue; and oxygen, red). The ChrR-CLD is shown as a molecular surface, colored pink except for residues that contact the ChrR-ASD, which are color coded as follows: carbon atoms, white; nitrogen, blue; and oxygen, red). Hydrogen bonding interactions (involving ChrR-ASD Glu22 and a network of hydrogen bonds around the ChrR-ASD-Zn²⁺ ligands His6 and His31) are denoted by yellow dashed lines. (B) Stereo view of contacts between the ChrR-ASD (purple carbon atoms) and CLD (pink carbon atoms) immediately surrounding the ChrR-ASD Zn²⁺ ligands His6 and His31.

Figure 5. Comparison of the ASDs of Rsp ChrR and Ec RseA

(A) The Rsp ChrR-ASD (purple) and Ec RseA-ASD (orange; Campbell et al., 2003), shown as backbone worms, are superimposed over the first three α helices (H1–H3). Rsp ChrR-ASD α carbons 9–19, 27–34, and 38v53 were aligned with Ec RseA-ASD 3–13, 18–25, and 29–44, yielding an rmsd of 1.4 Å over 33 atoms. (B) Alignment of the Ec σ^E/RseA-ASD and Rsp σ^E/ChrR complexes over α carbon positions of σ^E₂ only reveals the alignment of H4 from each anti-σ. (C) Structure-based sequence alignment between the ChrR and RseA ASDs. Amino acid similarities are highlighted in gray. The following residues are considered similar: VMLIA, DE, RK, and FYW. The secondary structure (α helices) of each protein is indicated above (Rsp ChrR-ASD, purple) or below (Ec RseA-ASD, orange). The ChrR-ASD Zn²⁺ ligands are indicated by asterisks.

Figure 6. Alignment and Secondary Structure Predictions for 27 Proposed ASD Sequences of Group IV Anti-σ Factors

An alignment based on sequence similarity for ASDs that display: (1) secondary structure, including transmembrane segments, and (2) projection of 3D structural elements across homologous sequences, using the Rsp ChrR-ASD (top) and Ec RseA (bottom) as endpoints. Secondary structure predictions for the ASD sequences using Jnet (Cuff and Barton, 1999) are shown as colored segments in the alignment, with α helices shaded in either in tan (Helices 1–3) or pink (Helix 4). Helix 3 of the STkin_Bs and Fpv_Pa sequences was not found by Jnet but was detected with PSIPRED (Jones, 1999). Any potential transmembrane helix is shown in gray, based on predictions with TMHMM (Krogh et al., 2001) and testing of some cases with PHDhtm (Rost et al., 1996). Bracketed numbers show the number of residues not shown in some parts of the alignment. The labeled helix boxes display the location of the Rsp ChrR-ASD α helices derived from the structure. Residues within conserved motifs are highlighted, with the ZAS motif (Cys/HisX_23–26HisX₃CysX₂Cys) in yellow and the FecR motif (TrpX₃AspX₂His) in blue. Residues with conservative conservation are printed in bold, colored letters, and a consensus is shown below. The 27 ASD sequences shown were extracted from a larger alignment of 1082 proteins (Table S1).