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. 2019 Mar 13;9:4346. doi: 10.1038/s41598-019-39554-y

Figure 6.

Figure 6

Chemical modification of the Cys30 residue of HlyU by CM14. (a) MS/MS spectrum of Cys30-modified peptide (RLQILC#MLHNQELSVGELCAK) from the CM14-treated HlyU protein. C# indicates the mass shift of C9H6O (# = +130.042 Da) by the cysteine modification. Both N- and C-terminal fragment ion series are represented as b and y series, respectively (e.g. b5, b6, b7… and y1, y2, y3…), and the annotated fragment ions are marked in the inserted peptide sequence. The observed precursor ion (monoisotopic m/z 862.440) in the inserted high resolution MS spectrum matched exactly with a theoretical m/z (862.439). (b) EMSA of the wild-type (WT), C30S mutant, or C96S mutant HlyU proteins and the radioactively-labeled PrtxA DNA complexes. The HlyU proteins (100 nM) were added to the probe DNA (5 nM) along with either 10% DMSO (control) or CM14 (10 μM) as indicated, and then the complexes were separated as described in Fig. 5. The cropped gel image is shown, and the full-length gel is presented in Supplementary Figure S8e. B, bound DNA; F, free DNA. (c) The hlyU mutants containing plasmids expressing wild-type or mutant HlyUs as indicated were grown along with CM14 (20 μM) or DMSO (control). The rtxA transcript levels in the total RNA of the cells were quantified by qRT-PCR and expressed using the transcript level of each group in the presence of DMSO as 1. Error bars represent the SD from biological triplicates. Statistical significance was determined by the Student’s t-test (*p < 0.05; ns, not significant). (d) Structural comparison of the CM14-treated HlyU (green, PDB code: 5ZNX) and the apo-HlyU (magenta, PDB code: 3JTH). The distances between the DNA-binding helices (𝛼4) are indicated. (e) Electron density map around Cys30 of the CM14-treated HlyU structure. The 2Fo–Fc (blue mesh) and the Fo–Fc (green mesh) maps are contoured at 1.5 σ and 4.4 σ, respectively. (f,g) Close-up views around Cys30 and Cys96 of the apo-HlyU (f) and the CM14-treated HlyU (g). The distances between sulfur atoms of the two cysteine residues are indicated.