Volume 28, Issue 4, Pages 652-664 (November 2007) Structural Mechanism of Organic Hydroperoxide Induction of the Transcription Regulator OhrR  Kate J. Newberry, Mayuree Fuangthong, Warunya Panmanee, Skorn Mongkolsuk, Richard G. Brennan  Molecular Cell  Volume 28, Issue 4, Pages 652-664 (November 2007) DOI: 10.1016/j.molcel.2007.09.016 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Structures of Reduced and Oxidized Xc OhrR (A) Structure of reduced OhrR is shown with one subunit colored magenta and the other subunit colored light pink. Residues Y36 and Y47 are shown as red sticks. Cysteines are shown as yellow sticks. (B) Structure of oxidized OhrR with one subunit colored teal and the other subunit colored light blue. The residues shown as sticks are colored as in (A). (C) Sequence and secondary structure alignment of the Bs and Xc OhrR proteins. Secondary structural elements are colored magenta for reduced Xc OhrR and teal for oxidized Xc OhrR; light blue and light pink boxes represent 310 helices. Reactive cysteine residues are colored yellow, and Y36 and Y47 are colored red. Shown in green is residue L17 that is strictly conserved in all OhrR protein sequences. Figures 1, 2, 3, 5 and 6 were made with PyMOL (DeLano, 2002). Molecular Cell 2007 28, 652-664DOI: (10.1016/j.molcel.2007.09.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Representative Electron Density for Reduced and Oxidized Xc OhrR Centered about Residues Y36-C22-Y47 2Fo − Fc electron density is shown in blue mesh and contoured to 1σ. Reactive tyrosines are shown as red sticks, and cysteine residues are shown as yellow sticks. (A) Reduced Xc OhrR is depicted as pink and magenta ribbons, and (B) oxidized Xc OhrR is depicted as teal and light blue ribbons. Molecular Cell 2007 28, 652-664DOI: (10.1016/j.molcel.2007.09.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Structural Changes upon Oxidation (A) The oxidized Xc OhrR dimer, shown as teal and light blue ribbons, has been superimposed on the reduced Xc OhrR dimer, which is depicted as magenta and light pink ribbons. Cysteines are shown as yellow sticks that emanate off yellow ribbons. The Cα backbone of residues 19–34 of helix α1 was used for the superposition. (B) Structural rearrangement of helix α6 upon oxidation. The superposition from (A) is shown for one subunit of the OhrR dimer. Color scheme is the same as in (A). (C and D) Key interactions between helix α5 and helix α2 in reduced (C) and oxidized (D) OhrR. Hydrogen bonds are depicted as black dashed lines, and important residues are shown as blue sticks. Reduced Xc OhrR is depicted as magenta and light pink ribbons in (C), and oxidized OhrR is depicted as teal and light blue ribbons in (D). Molecular Cell 2007 28, 652-664DOI: (10.1016/j.molcel.2007.09.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Induction of Wild-Type and Mutant OhrR Proteins by OHPs Background levels of expression in the absence of oxidants are shown as black bars. (A) tert-butyl hydroperoxide or (B) cumene hydroperoxide induction of the ohrR P1 promoter. The concentrations of each oxidant are shown within the graph. All assays were performed at least three times. Each error bar indicates the standard deviation of the averaged value. Molecular Cell 2007 28, 652-664DOI: (10.1016/j.molcel.2007.09.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Structural Changes Resulting from OHP Oxidation of OhrR (A) Rearrangement of tyrosines surrounding C22 upon OHP oxidation. Oxidized OhrR is colored teal and light blue, and reduced OhrR is colored magenta and light pink. Hydrogen bonds are shown as black dashed lines, and key residues are shown as sticks. (B) Steric clash of Y36 with α5 upon OHP oxidation of C22. van der Waals contacts are shown as magenta dashed lines for the reduced and teal dashed lines for the oxidized form. Key residues are shown as sticks. Molecular Cell 2007 28, 652-664DOI: (10.1016/j.molcel.2007.09.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Key Interactions Centered about Helix α2 in the Reduced and Oxidized Forms of Xc OhrR (A) van der Waals contacts made by L17 in the reduced OhrR structure are shown as dotted lines. Residues making direct and networked contacts to L17 are shown as blue sticks. The OHP sensor cysteine residue is shown as yellow sticks, and Y36 and Y47 are shown as red sticks. (B) Rearrangement of the L17 hydrophobic pocket and disorder of helix 1b upon oxidation. Residues interacting with L17 in the reduced form are shown in their new positions in the oxidized form. The side-chain color scheme is the same as in (A). Molecular Cell 2007 28, 652-664DOI: (10.1016/j.molcel.2007.09.016) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Putative OHP-Binding Pocket (A) The reduced OhrR dimer is shown as a surface representation with each subunit colored either magenta or light pink. Cumene hydroperoxide shown is modeled into the hydrophobic cleft and is shown as sticks with carbons colored cyan and oxygens in red. C22 is shown as a yellow surface, and Y36 and Y47 are shown as red surfaces at the bottom of the binding cleft. (B) A magnified view of the potential binding site for OHPs. Colors are the same as in (A) with labeled residues shown as sticks. Molecular Cell 2007 28, 652-664DOI: (10.1016/j.molcel.2007.09.016) Copyright © 2007 Elsevier Inc. Terms and Conditions