Significance: In gram-negative bacteria, OxyR is the master peroxide sensor that regulates the transcription of defense genes in response to a low level of cellular H ₂ O ₂ via a rapid kinetic reaction. In this study, we present the first, to our knowledge, full-length structures of peroxide-sensing transcription regulator OxyR together with an oxidation intermediate-mimicking structure. The structures show all of the structural features describing the tetrameric assembly and a bound H ₂ O ₂ molecule near the conserved cysteine. Combining the structural results, we reveal a step-by-step molecular mechanism for OxyR from H ₂ O ₂ sensing to structural changes for transcriptional activation. Our study provides a structural basis for potentially answering key questions about the role of the cysteine residue in other Cys-based sensors, even mammalian ones, in response to various oxidants.

Nuclear structure and function are governed by lamins, which are intermediate filaments that mostly consist of α-helices. Different lamin assembly models have been proposed based on low resolution and fragmented structures. However, their assembly mechanisms are still poorly understood at the molecular level. Here, we present the crystal structure of a long human lamin fragment at 3.2 Å resolution that allows the visualization of the features of the full-length protein. The structure shows an anti-parallel arrangement of the two coiled-coil dimers, which is important for the assembly process. We further discover an interaction between the lamin dimers by using chemical cross-linking and mass spectrometry analysis. Based on these two interactions, we propose a molecular mechanism for lamin assembly that is in agreement with a recent model representing the native state and could explain pathological mutations. Our findings also provide the molecular basis for assembly mechanisms of other intermediate filaments.

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.

Bo-Young Yoon, Ji-Hyun Yeom, Jin-Sik Kim, Si-Hyeon Um, Inseong Jo, Kangseok Lee, Yong-Hak Kim, and Nam-Chul Ha. Mol. Cells 2014;37:100-8. https://doi.org/10.14348/molcells.2014.2238