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. 2009 Aug 28;284(35):23517-24.
doi: 10.1074/jbc.M109.015826. Epub 2009 Jul 7.

Crystal structures of the reduced, sulfenic acid, and mixed disulfide forms of SarZ, a redox active global regulator in Staphylococcus aureus

Catherine B Poor  1 , Peng R Chen, Erica Duguid, Phoebe A Rice, Chuan He
Affiliations

Crystal structures of the reduced, sulfenic acid, and mixed disulfide forms of SarZ, a redox active global regulator in Staphylococcus aureus

Catherine B Poor et al. J Biol Chem. .

Abstract

SarZ is a global transcriptional regulator that uses a single cysteine residue, Cys(13), to sense peroxide stress and control metabolic switching and virulence in Staphylococcus aureus. SarZ belongs to the single-cysteine class of OhrR-MgrA proteins that play key roles in oxidative resistance and virulence regulation in various bacteria. We present the crystal structures of the reduced form, sulfenic acid form, and mixed disulfide form of SarZ. Both the sulfenic acid and mixed disulfide forms are structurally characterized for the first time for this class of proteins. The Cys(13) sulfenic acid modification is stabilized through two hydrogen bonds with surrounding residues, and the overall DNA-binding conformation is retained. A further reaction of the Cys(13) sulfenic acid with an external thiol leads to formation of a mixed disulfide bond, which results in an allosteric change in the DNA-binding domains, disrupting DNA binding. Thus, the crystal structures of SarZ in three different states provide molecular level pictures delineating the mechanism by which this class of redox active regulators undergoes activation. These structures help to understand redox-mediated virulence regulation in S. aureus and activation of the MarR family proteins in general.

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Figures

FIGURE 1.
FIGURE 1.
SarZ modification. A, NBD assay. SarZ Cys-S-NBD adduct peak appears at 420 nm, and the Cys-SO-NBD adduct appears at 347 nm. SarZ Cys13 to Ser mutant (C13S) and SarZ reacted with free cysteine or benzene thiol show no adduct peaks. B, reverse phase HPLC of SarZ reacted with benzene thiol, showing two components. C and D, molecular masses of components A (C) and B (D) determined by ESI-FTMS. The insets show the deconvoluted spectra.
FIGURE 2.
FIGURE 2.
Structures of reduced, sulfenic acid, and disulfide-modified SarZ, and sequence alignment. A, reduced SarZ structure with one monomer colored blue and the other colored gray. Cys13 is shown as yellow sticks. B, sulfenic acid form of SarZ structure with one monomer colored green and the other colored gray. Cys13-SOH is shown as yellow sticks. C, benzene thiol-modified SarZ structure with one monomer colored red and the other colored gray. Cys13 and BT residues are shown as yellow sticks. D, sequence alignment of SarZ with MgrA and BsOhrR. The secondary structural elements of SarZ-SOH, determined by the crystal structure, are shown as red tubes (helices) or green arrows (β-sheets). (SarZ-SH and SarZ-BT share the same secondary structural elements as SarZ-SOH, except for a shortening of helix α5 by one residue in SarZ-BT.) The residues are numbered according to the SarZ sequence. The reactive cysteine residues are highlighted by a yellow box. Residues forming the hydrogen bond network surrounding the cysteine are boxed in blue.
FIGURE 3.
FIGURE 3.
Electron density of the SarZ sulfenic acid modification. In SarZ-SOH, Cys13 is modeled as a thiol (A) or sulfenic acid (B). The positive electron density (blue) seen in A is satisfied by the sulfenic acid modification of B. Electron density maps of A are simulated annealing omit maps, with the oxygen of the sulfenic acid omitted. 2Fo − Fc maps, at 1.5 σ, are colored tan, and the Fo − Fc map, at 2.5 σ, is colored blue. The atoms are colored light blue (carbon), dark blue (nitrogen), red (oxygen), and yellow (sulfur).
FIGURE 4.
FIGURE 4.
Reactive Cys13 pocket. A, reduced Cys (SH). B, sulfenic acid Cys (SOH). Atoms are colored gray (carbon), red (oxygen), and yellow (sulfur). Water molecules are labeled W1 or W2 and shown as red spheres. Hydrogen bonds are shown as black dashed lines. A, Ser113′ to Tyr41′ 2.8 Å; Ser113′ to Tyr27′ 2.9 Å; Cys13 to Tyr27′ 3.3 Å; Cys13 to W1 3.9 Å; W1 to Tyr38′ 2.5 Å. B, Ser113′ to Tyr41′ 3.5 Å; Ser113′ to Tyr27′ 2.7 Å; Cys13-SOH Oδ to Tyr27′ 3.0 Å; Cys13-SOH Oδ to W1 2.7 Å; W1 to Tyr38′ 2.6 Å; W2 to Cys13-SOH Sγ 3.7 Å.
FIGURE 5.
FIGURE 5.
Structural implication of SarZ modification for binding to DNA. A, superimposition of the dimers of SarZ-SH (blue) and SarZ-SOH (green) highlights their similarity. B, superimposition of SarZ-SOH (green) and the BsOhrR-DNA (orange-purple) complex shows that SarZ-SOH is preconfigured for binding to DNA. C, superimposition of a single monomer of SarZ-SOH (green) and SarZ-BT (red). The other monomers of the dimers are aligned to highlight the changes in the monomers shown. D, superimposition of SarZ-SOH (green) and SarZ-BT (red) on DNA (purple) shows that SarZ-BT adopts a configuration unable to bind DNA. Alignment is along the monomers on the left to highlight the differences in the apposing monomers. Points of steric clash between SarZ-BT and DNA are circled. DNA is modeled based on the BsOhrR-DNA complex structure (25).
FIGURE 6.
FIGURE 6.
Cys13 pocket in SarZ-BT. A and B, BT-modified Cys13 of monomer C (A) and D (B). Atoms are colored gray (carbon), red (oxygen), and yellow (sulfur). Hydrogen bonds are shown as black dashed lines. A, Ser113′ to Tyr41′ 3.8 Å; Ser113′ to Tyr27′ 2.6 Å; Cys13-BT Sγ to Tyr38′ 3.3 Å. B, Ser113′ to Tyr41′ 2.9 Å; Ser113′ to Tyr27′ 2.5 Å. C, overlay of SarZ-SH (blue), SarZ-SOH (green), and SarZ-BT (red) showing the steric clash between Cys-BT and Phe117′ from either SarZ-SH or SarZ-SOH. In the SarZ-BT structure, Phe117′ is flipped away from the modification.
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