Evolution of sex-peptide in Drosophila

<p>The <i>Drosophila</i> sex-peptide (SP) has been identified as a seminal fluid component that induces post-mating responses (PMRs) in the inseminated females, such as inhibition of remating and stimulation of egg-laying. SP has been thought to play a central role in sexual conflict and sexually antagonistic co-evolution. Most of the sequenced <i>Drosophila</i> genomes contain SP orthologs, but their functions have been poorly characterized. Recently, we have investigated cross-species activity of <i>D. melanogaster</i> SP by means of injection into virgin females of other species. Among 11 species examined, SP response was observed in 6 species belonging to the <i>D. melanogaster</i> species group only. These species females express SP receptor (SPR) in their oviducts at relatively high levels, which was visualized by using a GFP-tagged SP. Furthermore, females of this species group responded to their own SP orthologs. However, females of the species outside the group did not respond to their own SP orthologs, even though all of them were potent inducers of SP-response in <i>D. melanogaster</i>. Our results suggested that the SP/SPR-mediated PMR was established in the lineage of the <i>D. melanogaster</i> species group.</p

Structural Basis of the γ-Lactone-Ring Formation in Ascorbic Acid Biosynthesis by the Senescence Marker Protein-30/Gluconolactonase

<div><p>The senescence marker protein-30 (SMP30), which is also called regucalcin, exhibits gluconolactonase (GNL) activity. Biochemical and biological analyses revealed that SMP30/GNL catalyzes formation of the γ-lactone-ring of l-gulonate in the ascorbic acid biosynthesis pathway. The molecular basis of the γ-lactone formation, however, remains elusive due to the lack of structural information on SMP30/GNL in complex with its substrate. Here, we report the crystal structures of mouse SMP30/GNL and its complex with xylitol, a substrate analogue, and those with 1,5-anhydro-d-glucitol and d-glucose, product analogues. Comparison of the crystal structure of mouse SMP30/GNL with other related enzymes has revealed unique characteristics of mouse SMP30/GNL. First, the substrate-binding pocket of mouse SMP30/GNL is designed to specifically recognize monosaccharide molecules. The divalent metal ion in the active site and polar residues lining the substrate-binding cavity interact with hydroxyl groups of substrate/product analogues. Second, in mouse SMP30/GNL, a lid loop covering the substrate-binding cavity seems to hamper the binding of l-gulonate in an extended (or all-trans) conformation; l-gulonate seems to bind to the active site in a folded conformation. In contrast, the substrate-binding cavities of the other related enzymes are open to the solvent and do not have a cover. This structural feature of mouse SMP30/GNL seems to facilitate the γ-lactone-ring formation.</p> </div

Behavioral response to tridecanoic acid in the OBP mutants.

<p>Behavioral responses of the <i>D. melanogaster</i> knockout flies for <i>Obp57d</i> and <i>Obp57e</i> to tridecanoic acid were examined by the oviposition site selection assay. Preference index (PI) = (<i>N_acid</i>−<i>N_cont</i>)/(<i>N_acid</i>+<i>N_cont</i>), where <i>N_acid</i> and <i>N_cont</i> are the number of eggs laid on tridecanoic acid-containing and control media, respectively. A total of 48 individuals were examined in the six independent replicates. (A) <i>w¹¹¹⁸</i>, (B) <i>Obp57d^KO</i>, (C) <i>Obp57e^KO</i>, and (D) <i>Obp57d+e^KO</i>.</p

Comparisons of the binding specificity among Dmel\OBP57d, Dmel\OBP57e, and Dpse\OBP57de.

<p>Binding affinity to various ligands was compared among the three OBPs using the quenching value (Q, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029710#pone-0029710-g002" target="_blank">Figure 2</a>). A higher 1/Q value means higher affinity to the ligand. Bars represent the means of three independent replicates, and error bars indicate standard error.</p

Overall structure of mouse SMP30/GNL.

<p>The structure is shown as a rainbow colored cartoon with N-terminus = blue and C-terminus = red. The divalent metal ion (labeled as M²⁺) located at the center of the structure is shown as an orange sphere.</p

p-values of the pairwise Wilcoxon rank sum test with adjustment by Holm's method for multiple comparisons for the results of the oviposition site selection assay.

<p>p-values of the pairwise Wilcoxon rank sum test with adjustment by Holm's method for multiple comparisons for the results of the oviposition site selection assay.</p

Active site structures of mouse and human SMP30/GNL.

<p>(<b>A</b>) Mouse SMP30/GNL in the substrate-free form, (<b>B</b>) the mouse SMP30/GNL–1,5-AG complex, (<b>C</b>) the human SMP30/GNL–1,5-AG complex, (<b>D</b>) the mouse SMP30/GNL–d-glucose complex, and (<b>E</b>) the mouse SMP30/GNL–xylitol complex. Lid loop residues of mouse SMP30/GNL and human SMP30/GNL are shown in purple and blue, respectively. Carbon atoms of ligand residues for the divalent metal ion (orange sphere) and those for substrate/product analogues are shown in green and yellow, respectively. Other carbon atoms are shown in white.</p

Expression and purification of recombinant OBPs.

<p>Bacterial cells before (−) and after (+) induction by IPTG, and purified protein (P) were analyzed by SDS-PAGE. (A) Dmel\OBP57d, (B) Dmel\OBP57e, and (C) Dpse\OBP57de. The expected size of the expressed OBPs is about 13 kDa.</p

Crystallographic data summary of human SMP30/GNL.

*<p>Values for the highest resolution shell are shown in parenthesis.</p

Top view of the substrate-binding cavity.

<p>Surface representation of (<b>A</b>) mouse SMP30/GNL, (<b>B</b>) DFPase, (<b>C</b>) Drp35, and (<b>D</b>) PON. Residues in the lid loop of mouse SMP30/GNL and the divalent metal ions (labeled as M²⁺) are shown in purple and orange, respectively. Structures of DFPase, Drp35, and PON are superposed onto mouse SMP30/GNL by the SSM fitting using the program Superpose <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053706#pone.0053706-Krissinel1" target="_blank">[27]</a> in the CCP4 program suite <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053706#pone.0053706-Collaborative1" target="_blank">[20]</a>, and all molecules are viewed from the same direction.</p