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, 112 (20), 6443-8

Structural Details of the OxyR Peroxide-Sensing Mechanism

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Structural Details of the OxyR Peroxide-Sensing Mechanism

Inseong Jo et al. Proc Natl Acad Sci U S A.

Abstract

OxyR, a bacterial peroxide sensor, is a LysR-type transcriptional regulator (LTTR) that regulates the transcription of defense genes in response to a low level of cellular H2O2. Consisting of an N-terminal DNA-binding domain (DBD) and a C-terminal regulatory domain (RD), OxyR senses H2O2 with conserved cysteine residues in the RD. However, the precise mechanism of OxyR is not yet known due to the absence of the full-length (FL) protein structure. Here we determined the crystal structures of the FL protein and RD of Pseudomonas aeruginosa OxyR and its C199D mutant proteins. The FL crystal structures revealed that OxyR has a tetrameric arrangement assembled via two distinct dimerization interfaces. The C199D mutant structures suggested that new interactions that are mediated by cysteine hydroxylation induce a large conformational change, facilitating intramolecular disulfide-bond formation. More importantly, a bound H2O2 molecule was found near the Cys199 site, suggesting the H2O2-driven oxidation mechanism of OxyR. Combined with the crystal structures, a modeling study suggested that a large movement of the DBD is triggered by structural changes in the regulatory domains upon oxidation. Taken together, these findings provide novel concepts for answering key questions regarding OxyR in the H2O2-sensing and oxidation-dependent regulation of antioxidant genes.

Keywords: OxyR; conformational change; hydrogen peroxide; reaction mechanism; transcription regulator.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overall structures of PaOxyR. (A) Two orthogonal views of the PaOxyR (C199D) FL protein (Left, bottom view; Right, side view). Each protomer is in magenta, cyan, yellow, or green, except for DBD2 (pale cyan) and DBD4 (dark green). The RDs of protomer 3 and protomer 1 are drawn in Cα tracing representation, but the intercysteine regions are in the ribbon diagram. Cys199 and Cys208 are displayed in stick representation. DNA-recognition helices α3 are labeled in the side view. (B) PaOxyR (WT) RD structure. Each protomer is in cyan or brown, except the intercysteine α-helical regions (magenta). The conserved cysteine residues are in stick representation. (C) PaOxyR (C199D) RD structure. Each protomer is colored in green or pink, except the intercysteine regions (residues 199–208 in magenta and residues 208–218 in orange). Asp199 and Cys208 are in stick representations. The disordered region is described in dotted line.
Fig. 2.
Fig. 2.
Two dimeric interfaces of the PaOxyR FL protein. (A) Two protomers are assembled by the dimeric interface at the RDs. The extended subunit is in green (RD) and dark green (DBD), whereas the compact subunit is in cyan (RD) and pale cyan (DBD). The hinge regions of an extended subunit are indicated by dotted red circles. The α-helices in the DBDs are labeled. The angles between the α4 linker helix of the DBD and the long axis to a line connecting two Cα carbons of residues 88 and 175 of the RD are 25° in the compact subunit and 155° in the extended subunit. (B) The dimeric interface at the DBDs between the compact subunit (cyan) and the extended subunit (magenta). The red rectangle indicates the interfaces for DBD–DBD and DBD–RD. (C) A close-up view of the red rectangular region of B. The residues that are involved in the DBD–DBD interaction are labeled in magenta or cyan, and the residues for the RD–DBD interaction in the compact subunits are in black.
Fig. 3.
Fig. 3.
H2O2-binding site. (A) The 2.0-Å-resolution structures are shown around the putative H2O2, superposed onto 2FoFc (blue) and FoFc (green) electron density maps contoured at 1.0 σ and 3.0 σ, respectively, when assuming an H2O2 molecule (Left) and when assuming a water molecule instead of H2O2 (Right). (B) Stereoview of the structure around the putative H2O2 and two water molecules. The broken lines indicate the polar interactions whose distances are within 3.3 Å. (C) A circular hydrogen-bonding network [Cys199-H2O2-w1-w2 (-Cys199)] and a hydrogen bond between His198 and w1. Sγ (blue circle) of Cys199 replaces the interaction of Asp199. The asterisk indicates a mutated residue.
Fig. 4.
Fig. 4.
Proposed mechanism for the S-hydroxylation of Cys199 as driven by bound H2O2. In the absence of H2O2, three water molecules are bound at the site near Cys199 by the residues His198 and Thr100 (step 1). The incoming H2O2 replaces a water molecule at the site, and H2O2 triggers the S-hydroxylation of Cys199 by the nucleophilic attack of the thiol group of Cys199 on the close OA atom of H2O2 (step 2). The resulting OBH acquires a proton transferred from the thiol of Cys199 via the two water molecules (w1 and w2) (step 2), leading to S-hydroxylation of Cys199 (step 3).
Fig. 5.
Fig. 5.
Putative intermediate structure given by the PaOxyR (C199D) RD. (A) Structural superposition of four protomers in the asymmetric unit of the PaOxyR (C199D) RD. The two-cysteine-containing regions are in magenta, orange, cyan, or blue. Asp199 and Cys208 are in stick representations. The disordered regions are drawn arbitrarily in broken lines. (B) Structural comparison of OxyR in different states. The PaOxyR RD in the reduced state (Left; cyan), the PaOxyR (C199D) RD (Middle; green), and the EcOxyR RD in the oxidized state (Right; purple) are displayed. (C) The interaction of Asp199 Oδ1. Asp199 Oδ1 forms hydrogen bonds with the backbone NH and/or carbonyl groups of Phe200 and Arg201, indicated by broken lines. In contrast, no interaction was observed with Asp199 Oδ2. S and O are in semitransparent blue circles in the Cys199-SOH structure. (D) Kinetic measurement of the thiol reactivity of PaOxyR RD variants [PaOxyR (C199D/C296S) and PaOxyR (C199S/C296S)] with DTNB. Both of the PaOxyR variants were at 35 µM concentration in 0.1 M Tris buffer (pH 7.5) containing 1% DMSO and 100 µM DTNB. The absorbance of the liberated TNB2− was measured at 412 nm. Five independent experiments were performed and the averaged lines are displayed (Left). The relative initial velocities were calculated from the slopes of the lines between 3 and 4 s (Right). The initial value of PaOxyR (C199S/C296S) is set to 1. The error bars indicate SE (n = 5).
Fig. 6.
Fig. 6.
Modeling studies of PaOxyR in the reduced and oxidized states. (A) Modeling of the DNA binding-competent structures of OxyR in the reduced (Left) and oxidized (Right) states. The crystal structure of PaOxyR is shown (Left). DBDs are colored in green and magenta, whereas RDs are colored in gray. Each α3 of the DBDs is indicated by number. (B) Schematic drawings of the OxyR tetramer in bottom views. The reduced form of OxyR (Left) and the oxidized form of OxyR (Right) are shown. Compact subunits are colored in green, and extended subunits are in orange. Hinge regions are indicated by black circles. Each DBD is numbered, and the regulatory domain is labeled. The distances between DBD1 and DBD3 are indicated by a double-headed arrow. The kink motion within the RD dimers (orange arrows) is converted to the inward motion of the DBD dimers (gray arrows).

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