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. 2019 Jun 13;10:2586. doi: 10.1038/s41467-019-10556-8

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© The Author(s) 2019

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. 3 — Molecular mechanism of SIP recognition by RopB. a Ribbon diagram of RopB–CTD dimer bound to SIP. Individual subunits of the dimer molecule are color-coded (blue and green). The 2Fo–Fc electron density map of SIP contoured at 1σ is shown. The SIP-binding pocket is boxed and labeled. The N- and C-termini of one subunit is marked as N and C, respectively. b Close up view of the interactions between RopB–CTD and SIP. SIP is shown as pink sticks and the eight amino acids of SIP are labeled. The SIP-interacting amino acid residues in RopB that are included in the mutational analyses from different subunits are colored in green and blue, respectively. The other SIP-contacting amino acid residues in RopB are colored in gray. The α-helices in RopB that form the SIP-binding pocket are labeled. c RopB–SIP-binding constants for synthetic SIP variants containing single alanine replacements at each position. d Analysis of the speB regulatory activity of synthetic SIP variants. The amino acid sequences of the synthetic peptides used in the experiment are shown in the inset. Scrambled peptide (SCRA) was used as a negative control. The SIP* mutant strain supplemented with DMSO was used as a reference and fold-changes in speB transcript levels relative to the reference are shown. P values (***P < 0.001, *P < 0.05) of the indicated samples were compared to SIP* mutant strain supplemented with WT SIP. e Western immunoblot analysis of secreted SpeB from indicated samples. Cell growth and synthetic peptide supplementation were performed as described in panel (d). Cell-free growth media (THY medium) were probed with anti-SpeB polyclonal rabbit antibody, and detected by chemiluminescence. The masses of molecular weight markers (M) in kilodaltons (kDa) are marked. Characterization of SIP mutant strains for speB gene transcript levels (f), secreted SpeB levels (g), and SpeB protease activity detected by casein plate assay (h). P values (****P < 0.0001) of the indicated strains were compared to SIP* mutant strain. P values were determined by t-test. Source data for panels (d) and (g) are provided as a Source Data file