Volume 27, Issue 5, Pages 793-805 (September 2007) A Conserved Structural Module Regulates Transcriptional Responses to Diverse Stress Signals in Bacteria  Elizabeth A. Campbell, Roger Greenwell, Jennifer R. Anthony, Sheng Wang, Lionel Lim, Kalyan Das, Heidi J. Sofia, Timothy J. Donohue, Seth A. Darst  Molecular Cell  Volume 27, Issue 5, Pages 793-805 (September 2007) DOI: 10.1016/j.molcel.2007.07.009 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Structure of σE/ChrR Complex (A) Ribbon diagrams (with α helices shown as cylinders) of the σE/ChrR complex. Regions of each protein are color coded: σE2, green; σE2-4 linker, cyan; σE4, 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 (σ4.1, orange; σ4.2, yellow). On the left is Ec σE4 from the Ec σE/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 σE4 from the Rsp σE/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 σ4.1 are aligned. Molecular Cell 2007 27, 793-805DOI: (10.1016/j.molcel.2007.07.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Zn2+ Coordination by the ChrR-ASD and Its Role in σE 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 σE-dependent transcription in vitro. The top panel shows the effects of adding indicated amounts of ChrR85, or full-length ChrR, on σE-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 σE-independent transcription (RNA1). Molecular Cell 2007 27, 793-805DOI: (10.1016/j.molcel.2007.07.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2007 27, 793-805DOI: (10.1016/j.molcel.2007.07.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

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. Molecular Cell 2007 27, 793-805DOI: (10.1016/j.molcel.2007.07.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

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 σE/RseA-ASD and Rsp σE/ChrR complexes over α carbon positions of σE2 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. Molecular Cell 2007 27, 793-805DOI: (10.1016/j.molcel.2007.07.009) Copyright © 2007 Elsevier Inc. Terms and Conditions

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/HisX23-26HisX3CysX2Cys) 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). Molecular Cell 2007 27, 793-805DOI: (10.1016/j.molcel.2007.07.009) Copyright © 2007 Elsevier Inc. Terms and Conditions