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. 2021 Jan 25;12:581. doi: 10.1038/s41467-020-20754-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Crystal structure of FA-activated HxlR protein/DNA complex (HxlR-WT-FA-DNA). Close-up view (right) shows the FA-triggered crosslinking reaction between residues Cys11 and Lys13 on helix α1. The 2F_o-F_c electron density map contoured at 1σ indicates that an intrahelical methylene bridge (pink) was formed between Cys11 and Lys13 upon FA treatment. b Crystal structure of HxlR protein in the absence of FA (HxlR-WT). Close-up view (right) shows the side-chains of residues Cys11 and Lys13 which face opposite directions. The distance between the thiol group on Cys11 and the amine group on Lys13 is 9.6 Å, indicating no interactions between these two residues. Superposed structures of HxlR-WT and HxlR-WT-FA-DNA (light green) showed obvious conformational change with FA-induced intrahelical crosslinking reaction. c MS-MS characterization of FA-induced methylene bridge between Cys11 and Lys13 on intact HxlR protein under living conditions. In the MS/MS spectrum of a peptide F9-K23, the fragment ions b2⁺ and y10⁺ correspond to the peptide fragments ₉FN₁₀ and ₁₄ELTLAVIGGK₂₃, respectively (observed m/z 262.12 and 1000.60), which verified the methylene bridge formation between Cys11 and Lys13 (+12 Da, highlighted by purple circles and the black line between them). d LC-MS analysis verifying the FA-induced methylene bridge between Cys11 and Lys13 on a peptide sequence derived from HxlR’s helix α1. Mutation of either Cys11 or Lys13 abolished the methylene bridge formation (+12 Da, the [M + C + H]⁺ ion). e Cys11 is essential for FA-enhanced DNA binding of HxlR. Determination of HxlR’s DNA-binding ability via EMSA showed that 0.6 mM FA was able to increase DNA-binding ability of HxlR-WT protein, whereas little change was observed for the HxlR-C11A mutant, in the presence of FA. f Flow cytometric analysis of the hxlAB-gfp reporter-harbored E. coli BW25113 cells expressing HxlR-WT or HxlR-C11A proteins with and without FA treatment. The addition of 0.6 mM FA increased the expression level of GFP in the bacterial strain expressing HxlR-WT, while little change was observed on the bacterial strain expressing HxlR-C11A, indicating that Cys11 is essential for FA-enhanced transcription activity of HxlR in vivo.