SAXS analysis of the ERK2:STEP complex.

<p>SAXS analysis of the ERK2:STEP complex.</p

The ERK2:STEP resting-state complex.

<p>(<b>A</b>) Constructs used in this study; (<b>B</b>) SAXS data (I(q) vs q) of the ERK2:STEP resting-state complex (black squares); error bars (grey lines). Error bars show the experimental error based on circular averaging of the 2D solution scattering data; theoretical scattering curve from calculated <i>ab initio</i> molecular envelope (red); <i>inset</i>, Guinier plots for samples at 1.0 mg/ml and 1.7 mg/ml; (<b>C</b>) The ERK2:STEP <i>ab initio</i> molecular envelope in two views rotated by 90° with the dimensions of the envelope.</p

Interaction of Kinase-Interaction-Motif Protein Tyrosine Phosphatases with the Mitogen-Activated Protein Kinase ERK2

<div><p>The mitogen-activation protein kinase ERK2 is tightly regulated by multiple phosphatases, including those of the kinase interaction motif (KIM) PTP family (STEP, PTPSL and HePTP). Here, we use small angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) to show that the ERK2:STEP complex is compact and that residues outside the canonical KIM motif of STEP contribute to ERK2 binding. Furthermore, we analyzed the interaction of PTPSL with ERK2 showing that residues outside of the canonical KIM motif also contribute to ERK2 binding. The integration of this work with previous studies provides a quantitative and structural map of how the members of a single family of regulators, the KIM-PTPs, differentially interact with their corresponding MAPKs, ERK2 and p38α.</p></div

Comparison of the P(r) functions of the MAPK:KIM-PTP resting state complexes.

<p>p38α:HePTP, maroon; p38α:PTPSL, red; p38α:STEP, dark blue; ERK2:HePTP, pink; ERK2:STEP, light blue (all p38α:KIM-PTP and ERK2:HePTP data were previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091934#pone.0091934-Francis1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091934#pone.0091934-Francis2" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091934#pone.0091934-Piserchio1" target="_blank">[17]</a>, but are shown here to provide a better comparison of the <i>P</i>(<i>r</i>) functions).</p

Regulatory protein-MAPK interaction strengths.

<p>The binding strengths of each KIM-PTP (KIM peptides and KIM-containing MKBDs (in orange font), light blue; KIMKIS peptides, light purple; proteins, light pink) to p38α and ERK2 represented from weakest to tightest (largest K_d to smallest K_d) binding affinity; ‘^†’ indicates values previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091934#pone.0091934-Francis1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091934#pone.0091934-Francis2" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091934#pone.0091934-Piserchio1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091934#pone.0091934-Kumar1" target="_blank">[18]</a>. KIM-PTP:MAPK complexes determined to be extended using SAXS written in normal text; KIM-PTP:MAPK complexes determined to be compact written in italics; *, compact nature of the ERK2-PTPSL complex determined by Balasu <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091934#pone.0091934-Balasu1" target="_blank">[24]</a> and corroborated by ITC measurements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091934#pone-0091934-t001" target="_blank">Table 1</a>).</p

BdcA cofactor is NADPH.

<p><b>A</b>) Thermal denaturation curves for BdcA. <b>B–D</b>) ITC data for BcdA with different cofactors (<b>B</b>, NADPH; <b>C</b>, NADP; <b>D</b>, c-di-GMP). The raw isothermal titration calorimetry data is shown in the upper panels, whereas the derived binding isotherm plotted against molar ratio of titrant are shown in the lower panels.</p

BdcA structural homologs as determined by DALI and FFAS.

<p>*model used for molecular replacement.</p><p>BdcA structural homologs as determined by DALI and FFAS.</p

Co-factor effects on BdcA thermal stability^*.

<p>*T_m shift data: Values represent means ± SD of twelve independent experiments</p><p>Co-factor effects on BdcA thermal stability^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105751#nt104" target="_blank">*</a>}.</p

Crystal data and data-collection statistics.

<p>Values in parentheses are for the highest resolution shell.</p>¥<p><i>R_merge</i> = Σ<i>_hkl</i>Σ_i |<i>I</i>_i(<i>hkl</i>) - <<i>I</i>(<i>hkl</i>)>|/Σ<i>_hkl</i>Σ_i<i>I</i>_i(<i>hkl</i>) where <i>I</i>_i(<i>hkl</i>) is the i^th observation of a symmetry equivalent reflection hkl. *Rfree was calculated using 5% of the reflections omitted from the refinement.</p><p>Crystal data and data-collection statistics.</p

Short-chain dehydrogenase/reductase elements of BdcA.

<p><b>A</b>) Sequence alignment of BdcA and homologous SDR members whose cofactors have been identified. Identical amino acids are highlighted in black and similar amino acids are highlighted in gray; α-helices are depicted as cylinders above the sequence alignment and the β-strands as arrows. Disordered loops are displayed as dotted lines. Conserved residues for NADP(H) binding are shown in lime and NAD(H) binding are shown in pink. The catalytic triad is highlighted in blue. <b>B</b>) Superposition of the residues that constitute the catalytic triad in BcdA (dark pink) and <i>E. coli</i> apo-FabG (gray, PDB:1I01; catalytically incompetent) and <i>E. coli</i> FabG bound to NADP+ (cyan, PDB:1Q7B; catalytically competent). <b>C</b>) Superposition of SDR family members that bind NADP(H), highlighting cofactor specificity residues. BdcA (dark pink, NADPH-specific), 4N5L (NADPH-specific, blue), 4DMM (NADP-specific, dark purple); all contain basic residues either in the Glycine-rich motif (BdcA, 4DMM) or after β2 (4N5L,). 4NBU (NADH-specific, coral) and 2AG5 (NAD-specific, orange) both contain an aspartic acid directly after the second β-strand. <b>D</b>) Predicted re-orientation of BdcA Arg16 upon NADPH binding. Overlay of BdcA (dark pink) with apo-SaFabG1 (yellow, PDB: 3OSU) and SaFabG1:NADPH (gold, PDB: 3SJ7; NADPH is depicted in dark gray).</p