<i>In vitro</i> adenylylation of MPT by PfuMoaB-WT and PfuMoaB-H3 variant.

<p>Adenylylation rates were determined for both proteins at 25, 35, 50, 65 and 80°C by monitoring formation of MTP-AMP over time. Initial velocities of PfuMoaB-WT and the H3 variant at different temperatures are depicted as solid and dotted lines, respectively. Error bars represent the standard deviation of data obtained in at least two independent experiments.</p

Structural Basis of Thermal Stability of the Tungsten Cofactor Synthesis Protein MoaB from <i>Pyrococcus furiosus</i>

<div><p>Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT) by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism <i>Pyrococcus furiosus</i>, the hexameric protein MoaB (PfuMoaB) has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15°C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50°C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface.</p></div

Biochemical characterization of the PfuMoaB-H3 variant in comparison to PfuMoaB-WT.

<p>(A) 15% Coomassie-Blue-stained SDS polyacrylamide gel showing 200 pmol of Ni-NTA-purified PfuMoaB-WT and PfuMoaB-H3. (B) Far-UV CD spectra of Ni-NTA purified PfuMoaB-WT (solid line) and PfuMoaB-H3 (dotted line). (C) Size exclusion chromatography of Ni-NTA purified PfuMoaB-WT and PfuMoaB-H3. 5 nmol of WT and 10 nm of PfuMoaB-H3 were applied on a Superdex 200 10/300 column. Peaks referring to the different oligomerization states of both proteins are labelled. Molecular masses were determined using protein standards. Elution of PfuMoaB-WT is shown as solid line, the PfuMoaB-H3 variant as dotted line. (D–E) SDS-PAGE of cross-linked PfuMoaB-WT and PfuMoaB-H3 with BS³ (D) and EDC (E). Samples without addition of cross-linkers were used as control (“–”). Observed oligomeric forms of both proteins are labelled. The cross-linked protein bands with a size corresponding to the trimers (designated with *) were further subjected to mass spectrometry analysis. (F) Differential scanning calorimetry of MPT-adenylyl-transferases. Melting curves of PfuMoaB-WT, PfuMoaB-H3, EcoMogA, EcoMoaB and AthCnx1G recorded by DSC. The maximum of each peak represents the respective <i>T</i>_m value. Average <i>T</i>_m values for each protein are summarized in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086030#pone-0086030-t002" target="_blank">Table 2</a>. Measurements were performed in duplicate for each experiment.</p

Active site of PfuMoaB-WT.

<p>Two PfuMoaB subunits at the hexamerization interface are shown as ribbon in green and grey, respectively. The conserved Asp56 residue coordinating Mg²⁺ (pink) is shown in sticks. MPT-AMP in the active site is derived from a superimposition with the structure of the PfuMoaB homologue <i>A. thaliana</i> Cnx1G (1UUY). The Mg²⁺-ion derived from a superimposition with the homologues sub-domain 3 of <i>E. coli</i> MoeA (1FC5) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086030#pone.0086030-Kuper1" target="_blank">[6]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086030#pone.0086030-Schrag1" target="_blank">[63]</a>.</p

Adenylylation of the cofactor intermediate MPT catalyzed by hexameric MoaB (<i>P. furiosus</i>) and trimeric MogA (<i>E. coli</i>).

<p>Adenylylation of the cofactor intermediate MPT catalyzed by hexameric MoaB (<i>P. furiosus</i>) and trimeric MogA (<i>E. coli</i>).</p

Multiple sequence alignment of MPT-adenylyl-transferases from different organisms.

<p>Corresponding MPT-adenylyl-transferases are abbreviated as follows: PfuMoaB, <i>Pyrococcus furious</i>; StoMoaB, <i>Sulfolobus tokodaii</i>; BceMoaB, <i>Bacillus cereus;</i> EcoMogA and EcoMoaB, <i>Escherichia coli</i>; TthMogA, <i>Thermus thermophilus</i>; AaeMogA, <i>Aquifex aeolicus</i>; <i>Arabidopsis thaliana</i>; HsaGephG, <i>Homo sapiens</i>. Secondary structure elements of PfuMoaB are shown. The conserved MPT-binding motif GGTG is highlighted with a red box, the conserved aspartate residue coordinating Mg²⁺- ion with a green box <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086030#pone.0086030-Kuper1" target="_blank">[6]</a>, residues of PfuMoaB α3-helix with a blue box. Highly conserved residues are depicted in white letters and black background; semi-conserved residues are shadowed in grey. Consensus threshold was set to 0.8. Sequences were aligned with Clustal Omega <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086030#pone.0086030-Sievers1" target="_blank">[62]</a>,and modified with BoxShade server (Swiss Institute of Bioinformatics).</p

Crystal structure of PfuMoaB.

<p>(A) Ribbon representation of PfuMoaB monomer, secondary structure elements, N- and C-termini are labelled; α-helices are coloured in cyan, β-sheets in magenta, 3₁₀-helices in green, loops in pink. (B) and (C) top and side view of the PfuMoaB hexamer, respectively. Subunits are shown in different colours. Zoom-in represent ionic interactions at the trimerization interface of PfuMoaB-WT. Residues mediating the contacts between subunits are shown in stick representation and are labelled. (D) Sulfate ion at the active site of PfuMoaB. The residues of the conserved Gly-Gly-Thr-Gly motif and the sulfate ion are shown superimposed with the experimentally phased electron density, contoured at 1 σ.</p

Depletion of Ecd or Prp8 causes apoptotic phenotypes in developing wings.

<p>(A–D) RNAi knockdown of <i>ecd</i> (B) and <i>prp8</i> (C) induced with the <i>dpp-Gal4</i> driver disrupted the regular pattern of the <i>dpp</i> expression domain (visualized by co-expression of <i>GFP</i>) in third-instar wing imaginal discs and caused appearance of apoptotic, Caspase 3 positive cells (B′, C′, white arrows). Expression of human Ecd (hEcd) averted the apoptotic phenotype caused by <i>ecd</i> RNAi (D, D′). (E–H) The intervein region corresponding to the <i>dpp</i> expression domain was reduced (double arrows) and the anterior crossvein (arrowhead) was lost in the wings of <i>dpp>ecd^RNAi</i> (F) and <i>dpp>prp8^RNAi</i> (G) adult flies. The <i>ecd</i> RNAi phenotype was rescued by expression of hEcd (H). Images in (A–D) are maximum-intensity projections of multiple confocal sections; panels A′ through D′ are higher-magnification, single sections from the corresponding images in (A–D). Scale bars are 50 µm (A–D), 20 µm (A′–D′), and 1 mm (E–H).</p

Subcellular localization of Ecd and the U5 snRNP proteins.

<p>(A) Brr2 was enriched in cell nuclei while Ecd, Prp8, Snu114, and Aar2 were detected in the cytoplasm. The Flag and Myc epitope-tagged proteins were expressed in S2 cells; mRFP::Prp8 and GFP::Ecd (bottom row) were co-transfected. (B) Inhibition of nuclear export with leptomycin B resulted in nuclear retention of the GFP::Ecd protein. Nuclei are stained with DAPI. All images are single confocal sections. Scale bars, 5 µm.</p

Loss of Ecd interferes with splicing of <i>spok</i> pre-mRNA.

<p>(A) Schematic of <i>Drosophila spok</i> and <i>phm</i> gene loci. Open and colored boxes represent untranslated and translated exons, respectively; black arrows point in the direction of transcription. Colored arrowheads mark the positions of primer pairs used to discriminate between pre-mRNA and mRNA species by qRT-PCR: yellow, in exons separated by an intron; red, within an intron; green and blue, spanning exon-intron boundaries of the first (EI1) and second (EI2) <i>spok</i> exon, respectively. (B) Pre-mRNA:mRNA ratios, determined by qRT-PCR with primers depicted in (A), were significantly elevated for <i>spok</i> but not for <i>phm</i> in third-instar <i>phm>ecd^RNAi</i> larvae and in <i>ecd¹</i> mutants at 29°C. (C, D) <i>spok</i> and <i>phm</i> pre-mRNA (C) and mRNA (D) levels decreased in <i>phm>ecd^RNAi</i> and <i>ecd¹</i> larvae. Levels of <i>α-tubulin 84B</i> mRNA did not change upon <i>ecd</i> RNAi and expression of <i>ecd</i> itself was unaffected by the <i>ecd¹</i> mutation (D). (E) RNAi knockdown of <i>prp8</i> in the PG diminished <i>spok</i> mRNA, whereas unspliced transcripts accumulated, causing the pre-mRNA:mRNA ratio to rise dramatically. (F) Expression of hEcd in the Ecd-deficient PG restored normal <i>spok</i> transcription and pre-mRNA splicing as judged from restored levels of pre-mRNA, mRNA, and their ratio. In all experiments, qRT-PCR was performed with total RNA from whole larvae 6 days AEL, and levels of <i>rp49</i> transcripts were used for normalization. The pre-mRNA:mRNA ratios in (B, E, F) were calculated from the normalized qRT-PCR data by dividing values obtained with intron primer sets (A, red triangles) or primers spanning exon-intron boundaries (green or blue triangles) with values obtained using mRNA-specific primers (yellow triangles). Data are mean ± S.E.M; <i>n</i>≥4; *<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001.</p