Matthew J. Wood

Structural basis for redox regulation of Yap1 transcription factor localization.

The ability of organisms to alter their gene expression patterns in response to environmental changes is essential for viability. A central regulator of the response to oxidative stress in Saccharomyces cerevisiae is the Yap1 transcription factor. Upon activation by increased levels of reactive oxygen species, Yap1 rapidly redistributes to the nucleus where it regulates the expression of up to 70 genes. Here we identify a redox-regulated domain of Yap1 and determine its high-resolution solution structure. In the active oxidized form, a nuclear export signal (NES) in the carboxy-terminal cysteine-rich domain is masked by disulphide-bond-mediated interactions with a conserved amino-terminal α-helix. Point mutations that weaken the hydrophobic interactions between the N-terminal α-helix and the C-terminal NES-containing domain abolished redox-regulated changes in subcellular localization of Yap1. Upon reduction of the disulphide bonds, Yap1 undergoes a change to an unstructured conformation that exposes the NES and allows redistribution to the cytoplasm. These results reveal the structural basis of redox-dependent Yap1 localization and provide a previously unknown mechanism of transcription factor regulation by reversible intramolecular disulphide bond formation.

Molecular mechanism of oxidative stress perception by the Orp1 protein.

In this study we investigated the molecular mechanism by which the Orp1 (Gpx3) protein in Saccharomyces cerevisiae senses and reacts with hydrogen peroxide. Upon exposure to H2O2 Orp1Cys36 forms a disulfide-bonded complex with the C-terminal domain of the Yap1 protein (Yap1-cCRD). We used 4-nitrobenzo-2-oxa-1,3-diazole to identify a cysteine sulfenic acid (Cys-SOH) modification that forms on Cys36 of Orp1Cys36 upon exposure to H2O2. Under similar conditions, neither Cys82 of Orp1Cys82 nor Cys598 of Yap1 forms Cys-SOH. A homology-based molecular model of Orp1 suggests that the structure of the active site of Orp1 is similar to that found in mammalian selenocysteine glutathione peroxidases. Proposed active site residues Gln70 and Trp125 form a catalytic triad with Cys36 in the Orp1 molecular model. The remainder of the active site pocket is formed by Phe38, Asn126, and Phe127, which are evolutionarily conserved residues. We made Q70A and W125A mutants and tested the ability of these mutants to form Cys-SOH in response to H2O2. Both mutants were unable to form Cys-SOH and did not form a H2O2-inducible disulfide-bonded complex with Yap1-cCRD. The pKa of Cys36 was determined to be 5.1, which is 3.2 pH units lower than that of a free cysteine (8.3). In contrast, Orp1 Cys82 (the resolving cysteine) has a pKa value of 8.3. The pKa of Cys36 in the Q70A and W125A mutants is also 8.3, demonstrating the importance of these residues in modulating the nucleophilic character of Cys36. Finally, we show that S. cerevisiae strains with ORP1 Q70A and W125A mutations are less tolerant to H2O2 than those containing wild-type ORP1. The results of our study suggest that attempts to identify novel redox-regulated proteins and signal transduction pathways should focus on characterization of low pKa cysteines.

Mutational Analysis To Define an Activating Region on the Redox-Sensitive Transcriptional Regulator OxyR

The OxyR transcription factor is a key regulator of the Escherichia coli response to oxidative stress. Previous studies showed that OxyR binding to a target promoter enhances RNA polymerase binding and vice versa, suggesting a direct interaction between OxyR and RNA polymerase. To identify the region of OxyR that might contact RNA polymerase, we carried out alanine scanning and random mutagenesis of oxyR. The combination of these approaches led to the identification of several mutants defective in the activation of an OxyR target gene. A subset of the mutations map to the DNA-binding domain, other mutations appear to affect dimerization of the regulatory domain, while another group is suggested to affect disulfide bond formation. The two mutations, D142A and R273H, giving the most dramatic phenotype are located in a patch on the surface of the oxidized OxyR protein and possibly define an activating region on OxyR.

A genetically encoded probe for the identification of proteins that form sulfenic acid in response to H2O2 in Saccharomyces cerevisiae.

It is widely known that reactive oxygen species (ROS), such as hydrogen peroxide, play important roles in cellular signaling and initiation of oxidative stress responses via thiol modifications. Identification of the targets of these modifications will provide a better understanding of the relationship between ROS and human diseases, such as cancer and atherosclerosis. Sulfenic acid is the principle product of a reaction between hydrogen peroxide and a reactive protein cysteine. This reversible post-translational modification plays an important role in enzyme active sites, signaling transduction via disulfide bond formation, as well as an intermediate to overoxidation products during oxidative stress. By re-engineering the C-terminal cysteine rich domain (cCRD) of the Yap1 transcription factor, we were able to create a genetically encoded probe for the general detection and identification of proteins that form sulfenic acid in vivo. The Yap1-cCRD probe has been used previously in the identification of proteins that form sulfenic acid in Escherichia coli. Here we demonstrate the successful use of the Yap1-cCRD probe in the identification of proteins that form sulfenic acid in response to hydrogen peroxide in Saccharomyces cerevisiae.