Crystal Structures of the Reduced, Sulfenic Acid, and Mixed Disulfide Forms of SarZ, a Redox Active Global Regulator in Staphylococcus aureus

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.

Staphylococcus aureus CymR Is a New Thiol-based Oxidation-sensing Regulator of Stress Resistance and Oxidative Response

“…However, after S-thiolation, their DNA binding ability is dramatically decreased (K d increases to 800 nM), which indicates that S-thiolation significantly alters the DNAbinding domain structure of CymR. This behavior is similar to the OhrR type proteins and has already been confirmed by a thiol-modified structure of SarZ, in which a dramatic conformational change was induced by S-thiolation (30).…”

“…However, in the OhrR type proteins, the sole cysteine is located toward the end of the first ␣ helix at the dimerization domain. First, oxidization and thiolation affect the conformation of the dimerization domain, which is transferred to the DNA-binding domains (29,30). Second, Cys-25 in CymR is quite exposed (Fig.…”

“…2B). Lys-20 and Arg-56 are less than 10 Å away from Cys-25, which may contribute to lower sulfhydryl pK a and increase its reactivity (30). In contrast, there are fewer basic residues surrounding the sole cysteine in OhrR, and the high reactivity of the active cysteine in OhrR was thought to be a result of the positive macrodipole of the first ␣ helix and hydrogen bonding to nearby Tyr-29 and Tyr-40, which helps to stabilize the negatively charged thiolate (29).…”

Protein cysteine phosphorylation of SarA/MgrA family transcriptional regulators mediates bacterial virulence and antibiotic resistance

“…S8C), with the DNA recognition helix α4 of SarZC13E displaced by 6 Å compared with that of the wild-type SarZ (Fig. S8D) (42). This observation suggests that Cys-phosphorylation of the SarA/MgrA family proteins induces changes at the dimeric interface, thereby modulating the DNA-binding properties of these proteins.…”

“…SarA, MgrA, and SarZ are dimeric proteins, in which the sole conserved Cys residue resides at the dimerization domain and is involved in hydrogen-bonding interactions with residues from the other monomer (31,42,43). To unveil how Cys-phosphorylation and the Cys-to-Glu substitution could impact DNA binding, we constructed initial structural models of wild-type Cys-phosphorylated SarZ and SarZC13E based on the crystal structure of SarZ in the Protein Data Bank (PDB ID: 3HSE) as described in SI Experimental Procedures.…”