title: Tetramers are the activation-competent species of the HOCl-specific transcription factor HypT.
abstract: Hypochlorous acid (HOCl) is an important component of the immune system and is produced by neutrophils to kill invading microorganisms. The transcription factor HypT is specifically activated by HOCl by methionine oxidation and protects Escherichia coli cells from the detrimental effects of HOCl. HypT forms dodecameric ring-like oligomers. Binding of HypT to DNA induces dissociation of the dodecamers into dimers and tetramers, thus forming the DNA-binding species. To dissect HypT dissociation, binding to DNA, and activation, we aimed to dissociate the dodecamers independently of DNA and to analyze HOCl-dependent activation in vitro. We found that HypT dodecamers dissociated into tetramers in the presence of l-arginine and NaCl, which was reversible upon dilution of the additive. Making use of the reversible dissociation, we generated mixed assemblies consisting of wild-type and mutant HypT subunits and determined that mutant subunits with reduced thermal stability were stabilized by wild-type HypT in the mixed assembly. HypT tetramers, as present at high NaCl concentrations, were stabilized against thermal unfolding and aggregation triggered by high HOCl concentrations. Importantly, in vitro activation by HOCl of HypT tetramers was completed within 1 min, whereas activation of dodecamers required 1 h for completion. Furthermore, activation of HypT tetramers required stoichiometric amounts of HOCl instead of an excess of HOCl, as observed for dodecamers. This supports the idea that small HypT oligomers are the activation-competent species, whereas the dodecamers are a storage form. Our study reveals the importance of the dynamic oligomeric state for HypT activation by HOCl.
authors.affiliation: From the Center for Integrated Protein Science Munich (CiPS), Department Chemie, Technische Universität München, 85747 Garching, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Drazic
authors.forename: Katharina M
authors.initials: KM
authors.lastname: Gebendorfer
authors.forename: Stefanie
authors.initials: S
authors.lastname: Mak
authors.forename: Andrea
authors.initials: A
authors.lastname: Steiner
authors.forename: Maike
authors.initials: M
authors.lastname: Krause
authors.forename: Alexander
authors.initials: A
authors.lastname: Bepperling
authors.forename: Jeannette
authors.initials: J
authors.lastname: Winter
mesh_terms: D001120:Arginine / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D002942:Circular Dichroism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005454:Fluorescence Polarization; D006997:Hypochlorous Acid / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D009154:Mutation; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects*; D055550:Protein Stability / Q000187:drug effects; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
chemical_list: D029968:Escherichia coli Proteins; C550912:HypT protein, E coli; D012097:Repressor Proteins; D012965:Sodium Chloride; D006997:Hypochlorous Acid; D004247:DNA; D001120:Arginine; D008715:Methionine
keywords: Bacteria; DNA-Protein Interaction; DNA-binding Protein; Methionine; Molecular Biology; Oxidative Stress; Protein Assembly; Protein Structure; Reactive Oxygen Species (ROS); Transcription Factors