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. 2015 Sep 20;23(9):747-54.
doi: 10.1089/ars.2015.6346. Epub 2015 Jun 22.

Does the Transcription Factor NemR Use a Regulatory Sulfenamide Bond to Sense Bleach?

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Free PMC article

Does the Transcription Factor NemR Use a Regulatory Sulfenamide Bond to Sense Bleach?

Michael Jeffrey Gray et al. Antioxid Redox Signal. .
Free PMC article

Abstract

Reactive chlorine species (RCS), such as hypochlorous acid (i.e., bleach), are antimicrobial oxidants produced by the innate immune system. Like many redox-regulated transcription factors, the Escherichia coli repressor NemR responds to RCS by using the reversible oxidation of highly conserved cysteines to alter its DNA-binding affinity. However, earlier work showed that RCS response in NemR does not depend on any commonly known oxidative cysteine modifications. We have now determined the crystal structure of NemR, showing that the regulatory cysteine, Cys106, is in close proximity to a highly conserved lysine (Lys175). We used crystallographic, biochemical, and mass spectrometric analyses to analyze the role of this lysine residue in RCS sensing. Based on our results, we hypothesize that RCS treatment of NemR results in the formation of a reversible Cys106-Lys175 sulfenamide bond. This is, to our knowledge, the first description of a protein whose function is regulated by a cysteine-lysine sulfenamide thiol switch, constituting a novel addition to the biological repertoire of functional redox switches.

Figures

<b>FIG. 1.</b>
FIG. 1.
The crystal structure of Escherichia coli NemRC106 only. (A) Shown is a schematic representation of NemR in two color schemes. Left panel: the two subunits of NemRC106 only dimer are colored in blue and yellow, respectively. Helices within one subunit are labeled αA–αI. Cys106 and Lys175 are colored red. Right panel: residues in one subunit are colored based on their sequence conservation. (B) Structural flexibility of the loop between helices αE and αF (EF loop). The four NemRC106 only subunit structures within the crystallographic asymmetric unit are superimposed and shown as white coils. The EF loops in the four subunits are colored in blue, pink, green, and orange, respectively. Cys106 and Lys175 are colored in red. Part of the loop structure in two subunits (green and orange) is not defined due to poor electron density. (C) Cys106 and Lys175 are in close proximity. Side chains of Cys106 and Lys175 as observed in the asymmetric unit are shown as stick models and colored using the following scheme: carbon (white), nitrogen (blue), and sulfur (yellow).
<b>FIG. 2.</b>
FIG. 2.
Oxidation reduces the free amine content of NemRC106 only. Oxidized and reduced NemR variants (1 ml, 50 μg ml−1) were mixed with fluorescamine (0.334 μl, 3 mg ml−1 in acetone) and fluorescence was measured, indicating the proportion of accessible lysine side chains in each sample.
<b>FIG. 3.</b>
FIG. 3.
Mass spectra of oxidized NemR are consistent with the formation of a cysteine 106–lysine 175 sulfenamide bond. (A) To investigate modifications of Cys106 and Lys175 upon NCT oxidation, we blocked any unmodified cysteine 106 with IAM in reduced and oxidized NemRC106 only and examined tryptic peptides both before and after DTT reduction. (B) Normalized LC-MS spectra revealed a reduction of the 957.48 Da IAM-modified Cys106-containing peptide (+3 charged) in NCT-treated NemRC106 only. (C) Conversely, the 938.47 Da Cys106-containing peptide (+3 charged) without IAM modification could be released by DTT reduction from a digest of IAM-treated NCT-oxidized NemRC106 only. (D) Predicted results of tryptic digestion of the Lys175-containing region of NemRC106 only with and without NCT oxidation. DTT reduction should have no effect on an unmodified lysine, while it should reduce a sulfenamide bond. (E) NCT treatment of NemRC106 only makes the potential cleavage site, Lys175, less accessible to trypsin and decreases the abundance of the 1109.57 Da Lys175-containing peptide (+3 charged). (F) DTT reduction of a tryptic digest of IAM-treated NCT-oxidized NemRC106 only increases the amount of the 1228.32 Da Lys175-containing peptide (+3 charged) without tryptic cleavage at Lys175. The predicted Cys106-Lys175 covalently linked peptide was not detected in any sample. However, this peptide is very large (6.1 kDa) and may not be easily detectable with LC-MS. DTT, dithiothreitol; IAM, iodoacetamide; LC-MS, liquid chromatography/mass spectrometry; NCT, N-chlorotaurine.
<b>FIG. 4.</b>
FIG. 4.
Disruption of lysine 175 prevents NemRC106 only DNA binding, but does not affect its oligomerization state. (A) EMSAs of the binding of NemRC106 only (0, 100, 200, 300, 400 nM) to nemR promoter DNA (PnemR, 10 nM) before and after treatment with NCT. To test the reversibility of oxidative inactivation, NCT-oxidized NemRC106 only was incubated with the thiol reductant, DTT (5 mM). Representative gels are shown along with quantification results (mean±SD, n=3). (B) EMSA of the binding of NemRC106 only, K175A to 10 nM PnemR DNA in the presence of 5 mM DTT (mean±SD, n=3). Concentration of NemR is plotted on a log2 scale. (C) Size exclusion chromatography was used to compare the oligomeric state of reduced and oxidized NemRC106 only and reduced NemRC106 only, K175A. Dotted lines indicate elution volume of protein standards of known molecular weight. EMSA, electrophoretic mobility shift assay.

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