title: Role of cysteines in the stability and DNA-binding activity of the hypochlorite-specific transcription factor HypT.
abstract: Reactive oxygen species are important components of the immune response. Hypochlorite (HOCl) is produced by neutrophils to kill invading microorganisms. The bactericidal activity of HOCl is due to proteome-wide unfolding and oxidation of proteins at cysteine and methionine residues. Escherichia coli cells are protected from HOCl-killing by the previously identified dodecameric transcription factor HypT (YjiE). Here, we aimed to unravel whether HOCl activates HypT directly or via a reaction product of HOCl with a cellular component. Bacterial viability assays and analysis of target gene regulation indicate that HypT is highly specific to activation by HOCl and that no reaction products of HOCl such as monochloramine, hydroxyl radicals, or methionine sulfoxide activate HypT in vivo. Surprisingly, purified HypT lost its DNA-binding activity upon incubation with HOCl or reaction products that oxidize HypT to form a disulfide-linked dimer, and regained DNA-binding activity upon reduction. Thus, we postulate that the cysteines in HypT contribute to control the DNA-binding activity of HypT in vitro. HypT contains five cysteine residues; a HypT mutant with all cysteines substituted by serine is aggregation-prone and forms tetramers in addition to the typical dodecamers. Using single and multiple cysteine-to-serine mutants, we identified Cys150 to be required for stability and Cys4 being important for oligomerization of HypT to dodecamers. Further, oxidation of Cys4 is responsible for the loss of DNA-binding of HypT upon oxidation in vitro. It appears that Cys4 oxidation upon conditions that are insufficient to stimulate the DNA-binding activity of HypT prevents unproductive interactions of HypT with DNA. Thus, Cys4 oxidation may be a check point in the activation process of HypT.
authors.affiliation: Center for Integrated Protein Science Munich at the Department Chemie, Technische Universität München, Garching, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Drazic
authors.forename: Amelie
authors.initials: A
authors.lastname: Tsoutsoulopoulos
authors.forename: Jirka
authors.initials: J
authors.lastname: Peschek
authors.forename: Jasmin
authors.initials: J
authors.lastname: Gundlach
authors.forename: Maike
authors.initials: M
authors.lastname: Krause
authors.forename: Nina C
authors.initials: NC
authors.lastname: Bach
authors.forename: Katharina M
authors.initials: KM
authors.lastname: Gebendorfer
authors.forename: Jeannette
authors.initials: J
authors.lastname: Winter
mesh_terms: D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006997:Hypochlorous Acid / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C550912:HypT protein, E coli; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; D006997:Hypochlorous Acid; D003545:Cysteine