Cys61 as an interface hub on CsdE exposed surface.

<p>(A) Opaque molecular surfaces of CsdE (white) with the position of Cys61 mapped out in yellow. From the top right corner and following a clockwise rotation, the following interaction surfaces are shown: TcdA (in pink), CsdA (persulfurated complex, in green), the two non-symmetric CsdE interaction surfaces (in cyan and in blue slate). (B) Close-up on the molecular surface of CsdE around Cys61 (yellow; labeled C61). The outlines of the interaction surfaces shown in (A) are drawn in thick line with the same color code; each area is labeled with the protein that occupies the respective surface area.</p

Structural comparison of CsdE along the proposed conformational change.

<p>(A) Superposition in cartoon with helices shown as cylinders of the CsdE free monomer from X-Ray (overlaid structures colored in green, white and red to highlight the movement, with the Cys61 Cα atom represented as a sphere), the CsdE monomer of dimer of the present study (pale blue) and CsdE monomer from the X-Ray structure of the (CsdA-CsdE)₂ complex (cyan). The CsdE free monomer is depicted over the ensemble-weighted maximally correlated mode contributing to the change in the selected distance (d[Cys61(Cα)–Val88(Cα)]). (B) Insight of the CsdE free monomer’s movement of the loop is shown with Cys61 side chain represented as balls and sticks.</p

Functional consequences of CsdE inactivation by disulfide bridge formation.

<p>A functional CsdA-CsdE sulfur mobilization system is depicted inside a cyan outline. CsdA, CsdE, and TcdA are represented as molecular surfaces. The tRNA molecules in the TcdA-tRNA complex are bead models derived from SAXS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186286#pone.0186286.ref041" target="_blank">41</a>]. CsdE is color coded in light cyan (CsdA unbound form) or bright cyan (bound to CsdA). CsdA subunits are colored in green and violet. TcdA subunits are shown in pale green and wheat. The tRNA bead models are in yellow. The inactivation of CsdE during oxidative stress conditions would likely lead to the impairment of the CsdA-CsdE downstream effector functions, the best known of which is the effect on ribosomal translation efficiency and fidelity through the TcdA-tRNA^ANN complex.</p

Thermal stability of FPS1S and FPS2 enzyme activity.

<p>(A) Activity of purified FPS1S (open symbols) and FPS2 (closed symbols) was measured after incubation at either 37°C (squares) or 45°C (circles) for the indicated time periods. (B) FPS activity in 16,000 <i>g</i> protein extracts from <i>fps1-1</i> (FPS2 activity, closed circles) and <i>fps2-1</i> (FPS1 activity, open circles) mutants was determined after incubation at 45°C for the indicated times. In both cases enzyme activities are expressed relative to the FPS activity values at time 0 min and the mean values and SE were calculated from three independent experiments. (C) Differential scanning fluorimetry (DSF) results plotted as change in fluorescence emission intensity (normalized to unity at its maximum) with increasing temperature (20–80°C). The FPS1S and FPS2 curves correspond to 6 µM enzyme.</p

Crystal structure of the CsdE dimer.

<p>(A) Top (left) and side (right) views of the disulfide-bridged CsdE dimer, shown in cartoon representation with the two chains in cyan and slate blue colors. Cys61 side chain and the disulfide bridge between them is represented in sticks, with the sulfur atoms in yellow and the carbon atoms in chain colors. (B) Zoom-in into the disulfide bridge holding together the CsdE dimer. The electron density map is a σ_A-weighted 2<i>DF</i>_O−<i>mF</i>_C map contoured at 1.2 σ in grey. (C) Angle between the vectors joining the center-of-mass of the disulfide bridge and those of monomers of the CsdE dimer during the simulation. The red line indicates the value determined from the X-ray structure.</p

Interaction network at the CsdE dimer interface.

<p>(A) The two CsdE monomers are shown in ribbon representation. Cys61 side-chain atoms are shown in yellow to mark its position with respect to the monomer-monomer interfaces. The structural elements of each monomer contacted by the opposite monomer are shown mapped in slate blue (top) or cyan (below). (B) Close-up around the disulfide bridge depicting the details of the interaction network that consolidates the dimer. Helices α7 are labeled H7 and β-strands β1, β2, and β3 are labeled as B1, B2, and B3, respectively, only in the top monomer (cyan). Cys61 is harbored in the small loop connecting β2 and β3. Interacting amino acid residues are represented in sticks (main and side chains) and the interactions are depicted by black dashed lines.</p

Characterization of <i>fps2-1</i> mutant lines harbouring <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> genes.

<p>(A) The expression of <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> was investigated using total RNA from 12-day-old seedlings of Arabidopsis wild-type, <i>fps2-1</i> and the indicated lines of the <i>fps2-1</i> mutant harbouring <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> chimeric genes. PCR products were electrophoresed in a 1% agarose gel. The size in bp of the amplified cDNA fragments corresponding to <i>FPS1mutdisp::FPS2</i> and <i>FPS1p::FPS2</i> (1088 bp) and <i>PP2A</i> genes (307 bp) is indicated on the right. Numbers on the left indicate the sizes in bp of DNA markers shown in lane M. (B) Western blot analysis of total FPS protein in 16,000 <i>g</i> extracts from seeds of plant lines indicated above (upper panel). The lower panel shows the Coomassie blue-stained electrophoretic protein patterns in the 35 to 50 kDa range of extracts used for FPS protein level determinations. Images show the results of one representative experiment. (C) FPS activity in the 16,000 <i>g</i> protein extracts used for Western blot analysis. FPS activity in mutants is expressed relative to that in the wild-type, which was assigned a value of 100. The mean values and SE were calculated from three independent experiments.</p

Expression in <i>E. coli</i> and purification of recombinant FPS1S and FPS2 proteins.

<p>(A) Total protein extracts from <i>E. coli</i> cells harbouring either pGEX-3X-NotI-FPS1 or pGEX-3X-NotI-FPS2 before (lanes 1 and 3) and after induction (lanes 2 and 4) of GST-FPS1S and GST-FPS2 expression with 0.4 mM IPTG for 6 hours at 22°C. (B) Soluble protein extracts of IPTG-induced <i>E. coli</i> cells harbouring either pGEX-3X-NotI-FPS1 (lane 1) or pGEX-3X-NotI-FPS2 (lane 2), and purified native FPS1S (lane 3) and FPS2 (lane 4) protein preparations after Glutathione-Sepharose 4B affinity column chromatography, proteolytic digestion with Factor Xa and protease removal. Arrows indicate the position of GST-FPS protein fusions and purified native FPS proteins. Molecular masses of standards (M) are indicated in kDa.</p

Trajectory followed by the Cys61 side chain of CsdE as it approaches CsdA for reaction.

<p>Three structures of CsdE are superimposed and represented in cartoons, with helices shown as cylinders. NMR CsdE is colored white, X-ray CsdE is in cyan, and the fully rearranged CsdE observed in the persulfurated (CsdA-CsdE)₂ complex is in green. Helices α6, α7, and, for persulfurated CsdE, α8’ and α8, are labeled in order to facilitate comparisons. The side chains of Cys61 and Glu62 are shown in sticks with carbon colors according to the corresponding CsdE structure and sulfur/oxygen atoms in CPK colors.</p

Homology modelling of FPS1S and FPS2 proteins and in silico <i>DD</i>G calculations.

<p>(A) Ribbon representation of the dimeric and monomeric structures of human FPS (PDB 2F7M), which was used to template the threading of Arabidopsis FPS1S and FPS2. The active site cleft is labelled and a bound phosphate ion is shown in sticks. (B) Sequence substitutions between FPS1S (top) and FPS2 (bottom) were mapped onto the ribbon structure (left monomer) or the molecular surface (right monomer) of the homology modeled dimers. Chemical character is colour coded as follows: red, acidic (Asp, Glu); blue, basic (Arg, Lys, His); green, polar (Ser, Thr, Asn, Gln, Tyr); orange, apolar (Met, Phe, Pro, Trp, Val, Leu, Ile, Ala). (C) Histogram of <i>DD</i>G (kcal/mol) upon single-site substitution calculated using Rosetta <i>DD</i>G application (top) or CC/PBSA (bottom). Mutations predicted to occur with a decrease in <i>DD</i>G are coloured green and those expected to increase <i>DD</i>G are coloured pink. Horizontal dashed lines at –1 to +1 kcal/mol bound the neutral area where <i>DD</i>G is supposed to contribute little to the overall stabilization or destabilization of the mutated protein.</p