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General information

OxyR is a protein that functions as a transcription factor. The OxyR we are analyzing is that of Escherichia Coli. In this perspective, the protein works by regulating the transcription of specific genes, being sensitive to H2O2. This transcription factor belongs to the LysR family, with 34 kDa, which forms homotetramers. Under this focus, the members of this family present a domain linked to DNA in their N-terminal region, called helix-loop-helix, a central domain responsible for recognizing H2O2 that is sensitive to the regulation signal and, finally, a domain in C-terminal region responsible for activation and mutimerization.

Structure

This protein its a transcription factor, at that time its shows two different structures when its reduced or oxidized. The reduced form monomer of the OxyR regulatory domain consists of two α/β domains that are linked by two interdomain strands. Residues in the middle of a folded domain form the majority of the N-terminal a/b domain. The C-terminal a/b Results and Discussion domain II is made of residues . The remaining C-terminal 30 Structure Determination residues come back to domain I to form an edge of the domain . The two domains exhibit a similar folding pattern consisting of a central b sheet flanked by helices and loops.

OxyR activation

The OxyR transcription factor is activated by the formation of an intramolecular disulfide bond. The Oxyr activation, by H2O2, begins with the oxidation of the Cys199 residue into a sulfenic acid intermediate, which causes destabilization of the Cys199 side chain in the hydrophobic binding pocket, due to its increased size and charge. This destabilization lead to expulsion of the side chain of Cys199 out of the interdomain pocket, resulting in a flexible loop around Cys199. These changes in the regulatory domain of OxyR allow the formation of an intramolecular disulfide bond between Cys199 and Cys208, separated by 17 Å in reduced form, which maintain OxyR in the active form.

Figure 1. Intermediates in the structural transitions of OxyR.

Protein activity in different forms

The OxyR transcription factor regulate the expression of genes involved in redox-homeostasis. OxyR protein is able to DNA-binding in its two different forms, oxidized and reduced. In your oxidized form, OxyR binds to four adjacent major grooves in DNA, while in the reduced form, OxyR binds to two pairs of adjacent major grooves separated by one helical turn (figure X). These two modes of binding allow OxyR to regulate different promoters, under different cell conditions. Both forms of OxyR act as repressors, regulating its own expression by oxyR transcription repression, while only oxidized OxyR is able to activate gene expression, inducing the expression of oxyS, hydroperoxidase I (katG), alkyl hydroperoxide reductase (ahpCF), glutathione reductase (gorA), glutaredoxin 1 (grxA) and other genes involved in redox-homeostasis, that protect the cell against oxidative stress.

Figure 2. Tetrameric Structure and a Plausible Model of DNA Interactions.

The tetramers observed in crystals of the reduced (A) and oxidized (B) forms are shown with the DNA positioned on the tetramers with plausible orientations. The tetramers are shown as ribbons (red, green, blue, and yellow for each monomer) and the model of DNA is represented as helical coils (cyan).

Purification and crystallization

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