Crystal Structure of Diedel, a Marker of the Immune Response of Drosophila melanogaster

Background: The Drosophila melanogaster gene CG11501 is up regulated after a septic injury and was proposed to act as a negative regulator of the JAK/STAT signaling pathway. Diedel, the CG11501 gene product, is a small protein of 115 residues with 10 cysteines. Methodology/Principal Findings: We have produced Diedel in Drosophila S2 cells as an extra cellular protein thanks to its own signal peptide and solved its crystal structure at 1.15 A ˚ resolution by SIRAS using an iodo derivative. Diedel is composed of two sub domains SD1 and SD2. SD1 is made of an antiparallel b-sheet covered by an a-helix and displays a ferredoxin-like fold. SD2 reveals a new protein fold made of loops connected by four disulfide bridges. Further structural analysis identified conserved hydrophobic residues on the surface of Diedel that may constitute a potential binding site. The existence of two conformations, cis and trans, for the proline 52 may be of interest as prolyl peptidyl isomerisation has been shown to play a role in several physiological mechanisms. The genome of D. melanogaster contains two other genes coding for proteins homologous to Diedel, namely CG43228 and CG34329. Strikingly, apart from Drosophila and the pea aphid Acyrthosiphon pisum, Diedel-related sequences were exclusively identified in a few insect DNA viruses of the Baculoviridae and Ascoviridae families. Conclusion/Significance: Diedel, a marker of the Drosophila antimicrobial/antiviral response, is a member of a small famil

Expression, purification, crystallization and preliminary X-ray analysis of the N-terminal domain of GNBP3 from Drosophila melanogaster

Crystals of the N-terminal domain of Gram-negative bacteria-binding protein 3 of D. melanogaster grown from PEG solutions are monoclinic (space group C2) and diffract to 1.7 Å resolution

Phylogenetic analysis of <i>Diedel</i>.

<p>(<b>A</b>) Genome localization of <i>Diedel</i> and related genes in drosophilids. Genes are named according to Flybase. The asterisk indicates that sequence has been been re-analysed and differs from the annotated one. For each <i>Drosophila</i> species are indicated the orientation and the position of the gene or on the chromosome (3R, X, 2) or on a scaffold. The precise position is indicated by the number at the beginning and the end of each base on the sequence given on fly base. When the distances are not too long, the position respect a scale (for pseudoobscura, persimilis and virilis). In the case of virilis, the gene GJ11856 is duplicated. No new ID has been proposed. (<b>B</b>) Phylogeny of Diedel-related molecules. The proteins are named according to Flybase for Drosophila species. IDs of the viral molecules can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033416#pone.0033416.s001" target="_blank">Text S1</a>. In red the melanogaster subgroup, in green the obscura group, in blue the repleta and the virilis groups, in purple the sequences from viruses. The sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033416#pone.0033416.s001" target="_blank">Text S1</a>.</p

Amino acid sequence alignment of Driedel and other homolous proteins.

<p>The Diedel protein of <i>Drosophila melanogaster</i> was aligned to homologous gene products from <i>Drosophila melanogaster</i> (CG34329) and <i>Pseudalatia unipuncta</i> granulovirus (PuGV). The numbering is that of Diedel in this study. Secondary structure elements, <i>i.e.</i> strands and helices, are indicated below the sequences as arrows and rectangles, respectively. Conserved residues are boxed, and strictly conserved residues are shown in white with a red background. Note that the level of sequence identity is much more higher in the SD2 sub-domain than in the SD1 sub-domain. The figure was generated with ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033416#pone.0033416-Gouet1" target="_blank">[36]</a>.</p

Special features of the Diedel structure.

<p>(<b>A</b>) Pro52 displays either <i>trans</i> or <i>cis</i> conformation. The structure obtained with the crystal form A (space group P212121) is colored in brown and that coming for the form B (space group P21212) is colored in cyan. The main structural difference is located in the loop 51–54 and is due to the <i>cis</i>-<i>trans</i> isomerization of the residue Pro52. (<b>B</b>) The loop 76–81 forms a conserved hydrophobic surface patch. The hydrophobic residues Ile78 and Phe79 located on the tip of the loop 76–81 are fully exposed. A network of hydrogen bonds involving the main chain nitrogen of residues 79 and 81 and the OE1 atom of the strictly conserved Asn77 maintains the loop in an extended conformation and contributes in the solvent exposure of the two hydrophobic residues.</p

Data collection and refinement statistics.

a<p><i>R</i>_merge = ∑_h∑_i|I_h,i−<i>_h|/∑_h∑_i I_h,i where <i>_h is the mean intensity of the symmetry-equivalent reflections.</i></i></p><i><i>b<p><i>R</i>_work = ∑_h∥F_o|−|F_c∥/∑_h|F_o| where F_o and F_c are the observed and calculated structure factor amplitudes, respectively, for reflection h.</p>c<p><i>R</i>_free is the <i>R</i> value for a subset of 5% of the reflection data, which were not included in the crystallographic refinement.</p></i></i

Overall crystal structure of Diedel.

<p>(<b>A</b>) Overall structure of Diedel. The structure is colored according to its secondary structure elements: α-helices (blue), 3₁₀ helices (pink), β-strands (green), and loops (brown). The 10 cysteine residues involved in five disulfide bridges are displayed in yellow and labelled. The N and C terminus residues are mentioned. The figure was generated with PyMOL (<a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>). (<b>B</b>) Topology diagram of Diedel. The color code for the secondary structure elements is similar to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033416#pone-0033416-g001" target="_blank">figure 1A</a>. Diedel is composed of two sub domains named SD1 and SD2. While SD1 belongs to the ferredoxin-like fold family, SD2 display a quite original fold highly reticulated with four disulfide bridges.</p