1. Volume 23, Issue 8, Pages 967-977 (August 2016)
Activity-Based Profiling Reveals a Regulatory Link between Oxidative Stress and Protein Arginine Phosphorylation 
Jakob Fuhrmann, Venkataraman Subramanian, Douglas J. Kojetin, Paul R. Thompson 
Cell Chemical Biology 
Volume 23, Issue 8, Pages (August 2016)
DOI: /j.chembiol
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2. Cell Chemical Biology 2016 23, 967-977DOI: (10. 1016/j. chembiol. 2016
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3. Figure 1 Inhibitors Targeting the Protein Arginine Phosphatase YwlE
(A) YwlE catalyzed dephosphorylation of pArg. The negatively charged phosphoryl group is highlighted in red, whereas the positively charged guanidinium group is shown in blue (see also Figure S1).
(B) Structure of synthesized compounds.
(C) Lineweaver-Burk plot depicting the mode of inhibition for SO3-amidine on YwlE activity using different inhibitor concentrations. Data are presented as mean ± SEM.
(D) Comparing the specificity of compound 9 for the inhibition of the arginine phosphatase YwlE with the closely related tyrosine phosphatase YfkJ. Data are presented as mean ± SEM (see also Figures S2 and S3).
Cell Chemical Biology  , DOI: ( /j.chembiol )
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4. Figure 2 Structure of SO3-Amidine-Ahx-BpA 9 Bound YwlE
(A) Left: overlay of 2D [1H,15N] HSQC NMR spectra of 15N,13C-labeled apo-YwlE (black) and [15N,13C]YwlE bound to SO3-amidine-Ahx-BpA (9) (orange). Right: inhibitor-induced changes in the NMR data mapped on the structure of YwlE; the substrate binding pocket is marked by a circle or line. Spheres mark residues substantially affected by inhibitor binding, including residues with large chemical-shift perturbations (blue), or residues with NMR peaks that are only present in the inhibitor bound (red) or apo structure (orange).
(B) Quantitation of chemical-shift perturbation (CSP) reveals residues affected by inhibitor binding. Color coding as described in (A).
(C) Molecular docking, using AutoDock v.4.2.5, of 9 (cyan) into YwlE (PDB: 4KK3, stripped of bound phosphate), which is illustrated as electrostatic surface representation. The right panel highlights a close-up view of the YwlE active site with the bound inhibitor (cyan). Polar contacts <3.5 Å are represented as dashed lines (see also Figure S4).
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5. Figure 3 Photo-Labeling of YwlE Using Rho-SO3-Amidine 10
(A) Structure of Rho-SO3-amidine (10).
(B) Photo-labeling of YwlE using photo-probe 10 in the presence and absence of H2O2, the competitive inhibitor SO3-amidine, and the specific oxidizing agent SeCN-amidine, which induces a disulfide bridge within the active site of YwlE. The fluorescence image is on top and the Coomassie-stained gel (which is below the fluorescent image) shows equal protein-loading levels (see also Figure S5).
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6. Figure 4 Functional and Structural Effects of YwlE Oxidation
(A–C) Mass spectrometric analyses of YwlE (A), YwlE treated with H2O2 (B), and preoxidized YwlE incubated with DTT (C). The ESI-MS spectra including the mass (black) and the charge status (red) of the distinct YwlE species is shown as well as the deconvoluted intact mass of the respective protein samples (theoretical mass of YwlE B.su = 17,850.3 Da).
(D) Residual activity of YwlE after treatment with the indicated oxidative stress-inducing agents. The activity was also tested after dialysis followed by treatment with the reducing agent DTT. Data are presented as mean ± SEM (n = 3).
(E) The image depicts the active-site architecture of YwlE bound to PO4 (left, PDB: 4KK3), and a model structure of YwlE containing a disulfide (S-S) bond (right). The disulfide-linked YwlE model was generated based on the apo-YwlE structure (PDB: 1ZGG), where the two cysteine thiol groups are only 4.1 Å apart. After manual cysteine side-chain rotation, the model was refined by applying geometry restraints including bond distances, angles, planes, and non-bonded contacts using Coot v (Emsley et al., 2010).
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7. Figure 5 Regulation of Cellular YwlE in B. subtilis Cells
(A) Pulldown of the active form of YwlE using 11 (upper panel) from differentially stressed B. subtilis cells and immunoprecipitation of endogenous YwlE (lower panel) from the respective cell samples (see also Figure S6).
(B) Influence of various inducers of oxidative stress on the arginine phosphorylation activity of McsB in the presence of YwlE. Arginine phosphorylated McsB was monitored using anti-pArg antibody by dot-blot assay (upper panel). As a control the same blot was stripped and reprobed with anti-McsB antibody (lower panel).
(C) Transcriptional analysis of CtsR repressed clp gene expression in wild-type versus ΔmcsB B. subtilis cells analyzed by qRT-PCR. The control samples were isolated from cells grown under non-stress conditions. Data from three independent experiments are presented as mean ± SEM (*p < 0.05, **p < 0.01).
(D) Model of YwlE-mediated regulation of arginine phosphorylation. YwlE acts as an arginine phosphatase to reverse the kinase activity of the protein arginine kinase McsB. Under oxidative stress YwlE is inactivated, thereby leading to an increased level of arginine phosphorylation that is important for the cellular stress response.
Cell Chemical Biology  , DOI: ( /j.chembiol )
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