Structure of the Mouse Sex Peptide Pheromone ESP1 Reveals a Molecular Basis for Specific Binding to the Class C G-protein-coupled Vomeronasal Receptor

Exocrine gland-secreting peptide 1 (ESP1) is a sex pheromone that is released in male mouse tear fluids and enhances female sexual receptive behavior. ESP1 is selectively recognized by a specific class C G-protein-coupled receptor (GPCR), V2Rp5, among the hundreds of receptors expressed in vomeronasal sensory neurons (VSNs). The specific sensing mechanism of the mammalian peptide pheromone by the class C GPCR remains to be elucidated. Here we identified the minimal functional region needed to retain VSN-stimulating activity in ESP1 and determined its three-dimensional structure, which adopts a helical fold stabilized by an intramolecular disulfide bridge with extensive charged patches. We then identified the amino acids involved in the activation of VSNs by a structure-based mutational analysis, revealing that the highly charged surface is crucial for the ESP1 activity. We also demonstrated that ESP1 specifically bound to an extracellular region of V2Rp5 by an in vitro pulldown assay. Based on homology modeling of V2Rp5 using the structure of the metabotropic glutamate receptor, we constructed a docking model of the ESP1-V2Rp5 complex in which the binding interface exhibited good electrostatic complementarity. These experimental results, supported by the molecular docking simulations, reveal that charge-charge interactions determine the specificity of ESP1 binding to V2Rp5 in the large extracellular region characteristic of class C GPCRs. The present study provides insights into the structural basis for the narrowly tuned sensing of mammalian peptide pheromones by class C GPCRs.     

Queen Bee Pheromone Binding Protein pH-Induced Domain Swapping Favors Pheromone Release

In honeybee (Apis mellifera) societies, the queen controls the development and the caste status of the members of the hive. Queen bees secrete pheromonal blends comprising 10 or more major and minor components, mainly hydrophobic. The major component, 9-keto-2(E)-decenoic acid (9-ODA), acts on the workers and male bees (drones), eliciting social or sexual responses. 9-ODA is captured in the antennal lymph and transported to the pheromone receptor(s) in the sensory neuron membranes by pheromone binding proteins (PBPs). A key issue is to understand how the pheromone, once tightly bound to its PBP, is released to activate the receptor. We report here on the structure at physiological pH of the main antennal PBP, ASP1, identified in workers and male honeybees, in its apo or complexed form, particularly with the main component of the queen mandibular pheromonal mixture (9-ODA). Contrary to the ASP1 structure at low pH, the ASP1 structure at pH 7.0 is a domain-swapped dimer with one or two ligands per monomer. This dimerization is disrupted by a unique residue mutation since Asp35 Asn and Asp35 Ala mutants remain monomeric at pH 7.0, as does native ASP1 at pH 4.0. Asp35 is conserved in only ∼ 30% of medium-chain PBPs and is replaced by other residues, such as Asn, Ala and Ser, among others, thus excluding that they may perform domain swapping. Therefore, these different medium-chain PBPs, as well as PBPs from moths, very likely exhibit different mechanisms of ligand release or receptor recognition.     

Mating Group Size and Stability in Reindeer Rangifer tarandus: The Effects of Male Characteristics,Sex Ratio and Male Age Structure

In polygynous mating systems, males compete intensely for mates and may mate several females during a single reproductive season. Accordingly, factors influencing the ability of males to control a larger number of females during the breeding season can provide information on the processes underlying sexual selection. In ungulates, age, body mass and social rank are considered good predictors of the reproductive success of males, but how male age structure and sex ratio in the population influence mating group (MG) dynamics has received little empirical testing. Between 1996 and 2005, we manipulated male age‐ and sex structure and monitored MG dynamics in a reindeer (Rangifer tarandus) population. We investigated the influence of male characteristics, percentage of males and male age structure on MG size and stability. We found that males with higher social rank (that were also older and heavier) controlled larger MGs (therefore had greater mating opportunities) and had more stable MGs (corresponding to a higher ability to maintain and control females) than males of lower social rank. Moreover, MG size and MG stability decreased as the percentage of males in the population increased, most likely resulting from greater male–male competition and increased female movements. Male age structure did not influence MG stability. Given the positive relationship between mating success and MG size (and likely MG stability), frequent female movements and intense competition among males to control females seem to be the principal components of reindeer MGs dynamic.     

Isolation and Structure of the Sex Pheromone Inhibitor,iAM373, of Enterococcus faecalis

An Enterococcus faecalis plasmid, pAM373, has a high frequency of transfer in a liquid medium when induced by a recipient-produced sex pheromone, cAM373. The sex pheromone inhibitor against cAM373, termed iAM373, was isolated from a culture supernatant of E. faecalis harboring pAM377 (=pAM373::Tn917), and its structure was identified as a heptapeptide, H-Ser-Ile-Phe-Thr-LeuVal-Ala-OH.     

Structure and Mode of Peptide Binding of Pheromone Receptor PrgZ

We present the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and OppA) explains the high specificity of PrgZ for hydrophobic heptapeptides versus the promiscuity of peptide binding in the homologous proteins.     

性类固醇激素对去势和未去势雄鼠脑微管蛋白合成的影响

雌性激素在某些方面改变着神经功能。在雌性激素敏感的神经元中,雌二醇调节各种ＲＮＡ的表达,包括ｒＲＮＡ、神经多肽和神经传导物受体。这种影响可能伴有在基因产物合成方面的相应改变或翻译后修饰。最近的研究已经揭示雌性激素能改变轴突生长以及突触密度。在我们以前的实验中注意到性别对脑微管蛋白合成的影响,也发现雌性激素可刺激雄鼠脑微管蛋白合成。雌性激素可能是成年动物神经突生长的一种调节物。这种假设已经得到深入研究和证实;发现性激素对大脑皮质、海马、中脑和间脑神经元的形态和数量有着重要的影响,并改变神经突生长和突触的连接。实验分为去势（ＧＭ）和未去势（ＩＭ）两组。第一组（ＧＭ）为１月龄雄鼠,去势后１０天分为睾丸酮（ＧＭＴ）、雌二醇（ＧＭＥ）、雌二醇加孕酮（ＧＭＥＰ）三个治疗实验组和一个对照组（ＧＭＣ）。第二组（ＩＭ）为老年雄鼠,分为睾丸酮（ＩＭＴ）、雌二醇（ＩＭＥ）、雌二醇加孕酮（ＩＭＥＰ）三个治疗组和对照组（ＩＭＣ）。对照组注射芝麻油。处理结束,迅速断头,制备脑匀浆液。取上清液进行３Ｈ秋水仙碱标记反应,液闪计数,测定脑微管蛋白的生成量。实验结果如表一：微管蛋白每分钟的放射量在ＧＭ组中,ＧＭＥ比ＧＭＣ增加３７％～４３％,Ｇ     

Evidence of a Male Sex Pheromone in the Round Goby (Neogobius melanostomus)

The reproductive success of the round goby (Neogobius melanostomus), an invasive fish, may be mediated by the use of pheromones. We hypothesized that reproductive male (RM) round gobies release sex pheromone to which reproductive females (RF) respond. In this study, we compared behavioural and electrophysiological responses of reproductive and non-reproductive female round gobies to conspeci fic males. Results of behavioural experiments in the laboratory showed that RF spent significantly more time near the source of the male odour compared with odours from control water. However, RF did not distinguish between odours from non-reproductive male (non-RM) water and control water. Non-reproductive females (non-RF) were not attracted to odours released from RM or non-RM water. Results of electro-olfactogram (EOG) responses showed that both RF and non-RF discriminated between HPLC fractionated RM and non-RM odours. However, the EOG responses of RF were about eight-fold higher than non-RF exposed to RM odours. These findings confirm that RM round gobies release a pheromone signal that attracts RF. The results of this research may be useful in developing control strategy using natural pheromones to disrupt the reproductive behaviour of the invasive round goby and to curtail its effects on native species. An erratum to this article is available at .     

The Solution Structure,Binding Properties,and Dynamics of the Bacterial Siderophore-binding Protein FepB

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs.     

The Sex Pheromone of the Mediterranean Flour Moth

Lachrymatory factor synthase (LFS), an enzyme essential for the synthesis of the onion lachrymatory factor (propanethial S-oxide), was identified in 2002. This was the first reported enzyme involved in the production of thioaldehyde S-oxides via an intra-molecular H⁺ substitution reaction, and we therefore attempted to identify the catalytic amino acid residues of LFS as the first step in elucidating the unique catalytic reaction mechanism of this enzyme. A comparison of the LFS cDNA sequences among lachrymatory Allium plants, a deletion analysis and site-directed mutagenesis enabled us to identify two amino acids (Arg71 and Glu88) that were indispensable to the LFS activity. Homology modeling was performed for LFS/23–169 on the basis of the template structure of a pyrabactin resistance 1-like protein (PYL) which had been selected from a BLASTP search on SWISS-MODEL against LFS/23–169. We identified in the modeled structure of LFS a pocket corresponding to the ligand-binding site in PYL, and Arg71 and Glu88 were located in this pocket.     

视黄醇类结合蛋白的分类、结构及功能

视黄醇类结合蛋白(retinoids binding protein)是体内负责结合并转运维生素A的各种活性代谢物(视黄醇类)的一类蛋白质,主要包括血浆视黄醇结合蛋白(RBP)、胞内视黄醇结合蛋白(CRBP)、胞内视黄酸结合蛋白(CRABP)、胞内视黄醛结合蛋白(CRALBP)和光受体视黄醇类结合蛋白(IRBP)五大类。它们空间结构高度保守,分布广泛,功能多样,与多种疾病的发生发展关系密切。     

Solution Structure of the N-terminal RNP Domain of U1A Protein: The Role of C-terminal Residues in Structure Stability and RNA Binding

The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an α-helix that lies across the β-sheet, with residues Ile93, Ile94 and Met97 making contacts with Leu44, Phe56 and Ile58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the β-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135° to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from Ile93 and Ile94 to Ile58, Leu 41, Val62 and His10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.     

Time and Sex in the Male Mouse: Temporal Regulation of Infanticide and Parental Behavior

Infanticide is a violent but successful reproductive strategy found in many mammals, particularly rodents. In male house mice (Mus domesticus and M. musculus) the act of ejaculation provides a reliable neural signal for timing the birth of their offspring. However, a unique chronobiological aspect of this phenomenon is the extraordinary temporal latency that can occur between the stimulus (coital ejaculation) and its adaptive neural response (male mice cease killing pups and behave parentally toward them instead). Specifically, the inhibition of infanticide is often time delayed for many days after a male ejaculates, but virtually always occurs before or around the time his own sired offspring would be born 18-20 days later. Furthermore, infanticide spontaneously reemerges 50-60 days after mating. In CF-1 stock male mice this entire behavioral sequence is synchronized with the female's reproductive cycle, and occurs even in the total absence of social cues or changes in pituitary or gonadal hormones after mating. When entrained and mated at 22 h (lightdark 11:11) or 27 h (lightdark 13.5:13.5) T-cycles, photoperiodic cues appeared to synchronize this dramatic shift in behavior, because a sudden transition from pup killing to parenting was matched with the number of light/dark cycles experienced after ejaculation rather than the amount of real time experienced, suggesting a circadian timing link. Housing in constant light accelerated the postmating transition to parenting, whereas constant dark significantly delayed the transition to parenting, but still occurred by 3 weeks after mating. Most males tend to oscillate between infanticide and parental behavior for several days before locking in to constant parenting, regardless of lighting conditions. Variation in the time delay between ejaculation and the inhibition of infanticide was consistent within young individuals (<10 months of age), but in older males (> 18 months of age) the time interval between ejaculation and parenting was significantly prolonged and attenuated. Another unique aspect of this phenomenon is that variation among individuals in their timing and response to light cues is correlated with phenotypic variation in sex steroid exposure during late fetal development. So far, no simple physiological explanation can account for the neural mechanism triggered by ejaculation that coordinates these time-delayed behavioral changes toward pups.     

Syntheses of 2-Isopropyl-4,5-dihydrothiazole and 6-Hydroxy-6-methyl-3-heptanone,Pheromone Components of the Male Mouse,Mus musculus

Syntheses of 2-isopropyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, pheromone components of the male mouse, Mus musculus, were achieved to provide sufficient amounts of samples for biological studies.     

Application of a Sex Pheromone,Pheromone Analogs,and Verticillium lecanii for Management of Heterodera glycines

A mutant strain of the fungus Verticillium lecanii and selected bioregulators of Heterodera glycines were evaluated for their potential to reduce population densities of the nematode on soybean under greenhouse conditions. The bioregulators tested were the H. glycines sex pheromone vanillic acid and the pheromone analogs syringic acid, isovanillic acid, ferulic acid, 4-hydroxy-3-methoxybenzonitrile, and methyl vanillate. A V. lecanii-vanillic acid combination and a V. lecanii-syringic acid combination were also applied as treatments. Syringic acid, 4-hydroxy-3-methoxybenzonitrile, V. lecanii, V. lecanii-vanillic acid, and V. lecanii-syringic acid significantly reduced nematode population densities in the greenhouse tests. Results with vanillic acid, isovanillic acid, and ferulic acid treatments were variable. Methyl vanillate did not significantly reduce cyst nematode population densities in the greenhouse tests.     

Sequence-Specific Size, Structure, and Stability of Tight Protein Knots

Approximately 1% of known protein structures display knotted configurations in their native fold, but the function of these configurations is not understood. It has been speculated that the entanglement may inhibit mechanical protein unfolding or transport, e.g., as in cellular threading or translocation processes through narrow biological pores. Protein knot manipulation, e.g., knot tightening and localization, has become possible in single-molecule experiments. Here, we investigate tight peptide knot (TPK) characteristics in detail by pulling selected 3₁ and 4₁-knotted peptides using all-atom molecular dynamics computer simulations. We find that the 3₁- and 4₁-TPK lengths are typically Δl ≈ 47± 4 Å and 69 ± 4 Å, respectively, for a wide range of tensions (0.1 nN ≲ F ≲ 1.5 nN). The 4₁-knot length is in agreement with recent atomic force microscopy pulling experiments. Calculated TPK radii of gyration point to a pore diameter of ∼20 Å, below which a translocated knotted protein might get stuck. TPK characteristics, however, may be sequence-specific: we find a different size and structural behavior in polyglycines, and, strikingly, a strong hydrogen bonding and water trapping capability of hydrophobic TPKs. Water capture and release is found to be controllable by the tightening force in a few cases. These mechanisms result in a sequence-specific “locking” and metastability of TPKs, which might lead to a blocking of knotted peptide transport at designated sequence positions. We observe that macroscopic tight 4₁-knot structures are reproduced microscopically (“figure of eight” versus the “pretzel”) and can be tuned by sequence, in contrast to mathematical predictions. Our findings may explain a function of knots in native proteins, challenge previous studies on macromolecular knots, and prove useful in bio- and nanotechnology.     

Agrotis segetum雄蛾触角叶性信息素反应神经元对电刺激触角神经的反应

为探讨电刺激Agrotis segetum雄蛾触角神经是否可以作为MGC中神经元的识别手段,采用细胞内电生理记录方法,共记录34个对性信息素有反应的MGC神经元,并测试了其中12个神经元对性信息素刺激的反应,22个神经元对性信息素刺激和电刺激的反应。结果表明,MGC神经元对性信息素及电刺激的反应模式基本一致,为一种双相反应模式。两种刺激方式均能诱导出兴奋反应,电刺激得到的兴奋反应比由信息素刺激引起的要短;MGC神经元对两种刺激的超极化反应（抑制反应）幅度影响没有显著性差别,在电刺激实验的22个神经元上,超极化反应幅度和抑制时间都与神经元本身放电频率有一定的相关性。超极化反应是在LN参与下一定的神经回路对刺激所产生的反应而形成的。这提示两种刺激所作用的神经回路应是一致的,但从整个实验过程记录到的神经元情况来看,还须进一步结合形态学实验来验证电刺激触角神经作为MGC神经元的识别手段。     

Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein,Visinin-Like Protein 3

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca²⁺-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca²⁺ binding, characterize Ca²⁺-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca²⁺ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (K_D = 0.52 μM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca²⁺-bound VILIP-3, whereas Ca²⁺-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca²⁺-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C₁₄ methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.