Comparison of canonical and non-canonical binding sites.

<p>(<b>A</b>) Structure of the PLCδ (1MAI.PDB) in steel blue with bound Ins(1,4,5)P₃ in the canonical binding site, which is formed by β1-β2, β3-β4 and β6-β7 loop regions; (<b>B</b>) Cartoon representation of Slm1-PH in green, Ins(4)P is bound in the non-canonical binding site; (<b>C</b>) Structure of the β-spectrin PH domain (1BTN.PDB) in wheat color with bound Ins(1,4,5)P₃ again in the non-canonical binding site. (<b>D</b>) Superposition of the Slm1-PH (green) modeled with Ins(1,4,5)P₃ onto the β-spectrin PH domain (wheat) and (<b>E</b>) onto PLCδ (cyan) (<b>F</b>) LIGPLOT representation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036526#pone.0036526-Wallace1" target="_blank">[38]</a> showing residues involved in ligand binding in the non-canonical binding site (Slm1-PH and β-spectrin) and the canonical binding site (PLCδ). The conserved residues between PH domains of Slm1 and β-spectrin involved in ligand binding are highlighted in pale yellow.</p

Data collection and refinement statistics.

*<p>Highest resolution shell is shown in parenthesis.</p>a<p>beam line ID29 at European Synchrotron Radiation Facility at Grenoble in France.</p>b<p>PX beam line at Swiss Light Source, Paul Scherrer Institut 5232 Villigen PSI Switzerland.</p>c<p>Rmeas is the redundancy-independent merging R-factor (intensities) <i>R</i>meas  = (Σ<i>h</i>(<i>n</i>/(<i>n</i>-1))^0.5 Σ<i>j</i>|<i>Îh</i> -<i>Ihj</i>|)/(Σ<i>hjIhj</i>) with <i>Îh</i>  = (Σ<i>jIhj</i>)/<i>nj</i> Where <i>N</i> is the number of times a given observation has been observed (ie j = 1, n).</p>d<p>3% of the total reflections were excluded for cross-validation.</p

Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly, protein extract of embryos from germinated barley seeds was treated ±Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (−Trx) samples were then chemically reduced and labeled with the “light” (12C) and “heavy” (13C) ICAT reagent, respectively. The extent of Trx-mediated reduction was thus quantified for individual cysteine residues based on ratios of tryptic peptides labeled with the two ICAT reagents as measured by liquid chromatography coupled with mass spectrometry (LC-MS). A threshold for significant target reduction was set and disulfide targets were identified in 104 among a total of 199 identified ICAT-labeled peptides. Trx-reduced disulfides were found in several previously identified target proteins, for example, peroxiredoxin and cyclophilin, as well as from a wide range of new targets including several ribosomal proteins that point to a link between Trx h and translation. The catalytic cysteine in dehydroascorbate reductase constituted the most extensively reduced target suggesting that Trx h has an important role in the ascorbate-glutathione cycle

Superposition of Slm1-PH domain structures.

<p>(<b>A</b>) The different ligands (sulfate (violet), phosphoserine (salmon), Ins(4)P (green) and also the turned-over Ins(4)P (light blue)) are occupying the same non-canonical binding pocket, confirming the readily available large binding site of the Slm1-PH. (<b>B</b>) Stereo diagram of PtdIns(4,5)P₂ (atom colors) modeled in the non-canonical binding site of Slm1-PH (green) and overlaid onto the β-spectrin PH domain (wheat color). This shows the conservation of side chains that contact the ligands.</p

Structure-based sequence alignment of PH domains of Slm1, β-spectrin and PLCδ.

<p>The alignment was produced using the PROMALS3D program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036526#pone.0036526-Pei1" target="_blank">[39]</a> and corrected manually on the basis of the three-dimensional structure of the Slm1-PH. The matched-up sequences were from RCSB/PDB. The secondary structure elements revealed in the Slm1-PH crystal structure are shown above the sequence: β-sheets are shown by blue arrows and the C-terminal helix by an orange cylinder. The residues of Slm1-PH involved in Ins(4)P interaction are shown in red. The residues highlighted in yellow are aligned with the ligand-interacting residues of the Slm1-PH. Residues of different classes of PH domains that are involved in ligand binding are shown in ‘bold’. The R/KXR and R/KXW motifs for ligand binding in the canonical and non-canonical binding sites respectively, are shown in the black box. The structure-based sequence alignment showing overall conserved or semi-conserved residues are represented in grey. Sequences are grouped according to ligand binding in the non-canonical and canonical binding pockets.</p

Crystallographic screening for Slm1-PH domain ligands.

*<p>PS  =  phosphoserine.</p>*<p>Crystals of the same protein-ligand complex were obtained in different crystallization conditions.</p>*<p>FTY720  =  synthetic analog of sphingosine, currently studied as a potent immunosuppressive and immunomodulatory agent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036526#pone.0036526-Mansoor1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036526#pone.0036526-Zhang1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036526#pone.0036526-Hiestand1" target="_blank">[24]</a>; FTY720(P)  =  phophorylated FTY720.</p

Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly, protein extract of embryos from germinated barley seeds was treated ±Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (−Trx) samples were then chemically reduced and labeled with the “light” (12C) and “heavy” (13C) ICAT reagent, respectively. The extent of Trx-mediated reduction was thus quantified for individual cysteine residues based on ratios of tryptic peptides labeled with the two ICAT reagents as measured by liquid chromatography coupled with mass spectrometry (LC-MS). A threshold for significant target reduction was set and disulfide targets were identified in 104 among a total of 199 identified ICAT-labeled peptides. Trx-reduced disulfides were found in several previously identified target proteins, for example, peroxiredoxin and cyclophilin, as well as from a wide range of new targets including several ribosomal proteins that point to a link between Trx h and translation. The catalytic cysteine in dehydroascorbate reductase constituted the most extensively reduced target suggesting that Trx h has an important role in the ascorbate-glutathione cycle

Surface charge distribution.

<p>(<b>A</b>) The Slm1-PH provides a positively-charged cavity to interact with a negatively charged Ins(4)P molecule (in green). The back of the β1-β2 region (by rotating Slm1-PH by 180 degrees) shows a more positively charged region. In several structures of Slm1-PH determined in complex with ligand analogs, we built a phosphate group or full Ins(4)P at this additional positively charged site (see text for details). The Arg477 side chain that turns towards the canonical binding site is also shown in transparent stick format. (<b>B</b>) Stereo diagram of Slm1-PH (green) showing Arg477, Arg478, Lys480 and Lys483 on either side of the β1-β2 strands in the vicinity of the negatively charged residues of the β5-β6 loop of the neighboring molecule (cyan). Part of the β5-β6 loop is truncated for the clarity of the figure. The bound phosphate group is shown in stick representation at the back of the β1-β2 region towards the canonical binding site. We have modeled the natural ligand (DHS-1P in yellow) aligning with the phosphate position in this region. Ins(4)P is also shown in the non-canonical binding site.</p

Detailed stereo diagrams of bound ligands.

<p>(<b>A</b>) Interactions made by Ins(4)P (green) and the residues at the non-canonical binding site of Slm1-PH (also in green). The phosphate group of Ins(4)P is making contacts with the β1-β2 region (residues involved are shown in red). Residues critically involved in the interactions are labeled by the single-letter code and shown in stick representation (see text for details). (<b>B</b>) The Ins(4)P is shown in the experimental 2Fo-Fc electron density map at 1.2 sigma level. The water molecule (red) corresponds to P1 position of Ins(1,4,5)P when docked into the non-canonical binding site. Position of the Ins(4)P is oriented to maximize the clarity of the figure. (<b>C</b>) Similar interactions are shown here with the Ins(4)P molecule for which the phosphate position is seen on the opposite side of the binding pocket, making contacts with residues of β5-β6 (again in red). (<b>D</b>) Stereo representation of phosphoserine bound in the non-canonical binding site. The dashed lines shown between atoms represent hydrogen bonds between ligand(s) and the residues at the non-canonical binding site of Slm1-PH.</p

Overall structure of the Slm1-PH domain.

<p>(<b>A</b>) Ribbon representation of the 1.76 Å structure (green) showing that Slm1-PH folds into a seven-stranded β-sheet terminated with an α-helix. All secondary structure elements are labeled in yellow and black. Both canonical and non-canonical binding sites are also marked. (<b>B</b>) In the X-ray crystal structure the canonical binding site is partially occluded by the β5-β6 loop (in red) of the neighboring molecules (in yellow and violet) on both sides. The β1-β2 loops of all the molecules are shown in blue. All the loops in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036526#pone-0036526-g001" target="_blank">Figure 1B</a> have been smoothened using PyMOL for clarity. (<b>C</b>) Detailed view of the interactions between the β1-β2 loop and the β5-β6 loop of neighboring molecules. Primarily, the side-chain residues of the β5-β6 loop are making contact with the main-chain atoms of the β1-β2 loop region.</p