Significance Hypochlorous acid (HOCl) is a powerful oxidant that kills microorganisms. HypT has been identified as a HOCl-sensing transcription factor regulating several genes to enhance survival during HOCl stress in… Click to show full abstract
Significance Hypochlorous acid (HOCl) is a powerful oxidant that kills microorganisms. HypT has been identified as a HOCl-sensing transcription factor regulating several genes to enhance survival during HOCl stress in Escherichia coli. However, the structure and a detailed action mechanism of HypT have not yet been reported. In this study, we identified Salmonella Typhimurium HypT as a crucial factor in survival within macrophages and presented structures of HypT. The full-length structure shows interesting features describing a type of tetrameric assembly for the LysR family transcription regulator. The regulatory domain structures at various states give important clues to understanding the HOCl-sensing mechanism. Combining these results, we provided a molecular mechanism for HypT that explains its HOCl-sensing ability and structural changes. Hypochlorous acid (HOCl) is generated in the immune system to kill microorganisms. In Escherichia coli, a hypochlorite-specific transcription regulator, HypT, has been characterized. HypT belongs to the LysR-type transcriptional regulator (LTTR) family that contains a DNA-binding domain (DBD) and a regulatory domain (RD). Here, we identified a hypT gene from Salmonella enterica serovar Typhimurium and determined crystal structures of the full-length HypT protein and the RD. The full-length structure reveals a type of tetrameric assembly in the LTTR family. Based on HOCl-bound and oxidation-mimicking structures, we identified a HOCl-driven methionine oxidation mechanism, in which the bound HOCl oxidizes a conserved methionine residue lining the putative ligand-binding site in the RD. Furthermore, we proposed a molecular model for the oxidized HypT, where methionine oxidation by HOCl results in a conformational change of the RD, inducing a counter rotation of the DBD dimers. Target genes that are regulated by HypT and their roles in Salmonella were also investigated. DNase I footprinting experiments revealed a DNA segment containing two pseudopalindromic motifs that are separated by ∼100 bp, suggesting that only the oxidized structure makes a concomitant binding, forming a DNA loop. An understanding of the HypT-mediated mechanism would be helpful for controlling many pathogenic bacteria by counteracting bacterial HOCl defense mechanisms.
               
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