Efficient production of cordycepin by engineered Yarrowia lipolytica from agro-industrial residues.

Cordycepin, a nucleoside compound with a variety of biological activities, has been extensively applied in the nutraceutical and pharmaceutical industries. The advancement of microbial cell factories using agro-industrial residues provides a sustainable pathway for cordycepin biosynthesis. Herein, the cordycepin production was enhanced by the modification of glycolysis and pentose phosphate pathway in engineered Yarrowia lipolytica. Then, cordycepin production based on economical and renewable substrates (sugarcane molasses, waste spent yeast, and diammonium hydrogen phosphate) was analyzed. Furthermore, the effects of C/N molar ratio and initial pH on cordycepin production were evaluated. Results indicated that the maximum cordycepin productivity of 656.27 mg/L/d (72 h) and cordycepin titer was 2286.04 mg/L (120 h) by engineered Y. lipolytica in the optimized medium, respectively. The cordycepin productivity in the optimized medium was increased by 28.81% compared with the original medium. This research establishes a promising way for efficient cordycepin production from agro-industrial residues.

Acinetobacter tibetensis sp. nov., Isolated from a Soil Under a Greenhouse in Tibet.

A Gram-stain-negative, light yellow, aerobic, non-motile, short rod-shaped bacterium named strain Y-23T with iprodione-degrading capability was isolated from a soil under a greenhouse in Tibet, PR China. Strain Y-23T grew at 4-37 â„ƒ and pH 5.0-9.0 (optimum, 25 â„ƒ and pH 7.0) with 0-3% (w/v) NaCl (optimum, 0%). Phylogenetic analysis based on 16S rRNA gene and chromosome genome indicated that strain Y-23T formed a stable evolutionary branch with Acinetobacter tandoii DSM 14970T. The 16S rRNA gene similarity, digital DNA-DNA hybridization and average nucleotide identity values between strain Y-23T and Acinetobacter tandoii DSM 14970T were 98.31%, 43.2% and 91.2%, respectively. The genome size was 3.39 Mbp with a genomic DNA G+C content of 40.59 mol%. The predominant fatty acids were C18:1 ω9c, Summed feature 3 (C16:1 ω7c/C16:1 ω6c), C12:0, C12:0 3-OH and C16:0. The polar lipids were diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline, unidentified phospholipid, four unidentified aminophospholipids and two unidentified lipids. The isoprenoid quinone was Q-8 (19.43%) and Q-9 (80.57%). Based on phenotypic, phylogenetic, and genotypic data, strain Y-23T is considered to represent a novel species of the genus Acinetobacter, for which the name Acinetobacter tibetensis sp. nov. is proposed. The type strain is Y-23T (= CICC 25150T = JCM 35630T).

High-level de novo biosynthesis of cordycepin by systems metabolic engineering in Yarrowia lipolytica.

Cordycepin is a nucleoside antibiotic with various biological activities, which has wide applications in the area of cosmetic and medicine industries. However, the current production of cordycepin is costly and time-consuming. To construct the promising cell factory for high-level cordycepin production, firstly, the design and construction of cordycepin biosynthetic pathway were performed in Yarrowia lipolytica. Secondly, the adaptivity between cordycepin biosynthetic pathway and Y. lipolytica was enhanced by enzyme fusion and integration site engineering. Then, the production of cordycepin was improved by the enhancement of adenosine supply. Furthermore, through modular engineering, the production of cordycepin was achieved at 3588.59 mg/L from glucose. Finally, 3249.58 mg/L cordycepin with a yield of 76.46 mg/g total sugar was produced by the engineered strain from the mixtures of glucose and molasses. This research is the first report on the de novo high-level production of cordycepin in the engineered Y. lipolytica.

Metabolic engineering of Yarrowia lipolytica for improving squalene production.

The aim of this present research is to enhance the squalene production in Yarrowia lipolytica using pathway engineering and bioprocess engineering. Firstly, to improve the production of squalene, the endogenous HMG-CoA reductase (HMG1) was overexpressed in Y. lipolytica to yield 208.88 mg/L squalene. Secondly, the HMG1 and diacylglycerol acyltranferase (DGA1) were co-overexpressed, the derived recombinant Y. lipolytica SQ-1 strain produced 439.14 mg/L of squalene. Thirdly, by optimizing the fermentation medium, the improved titer of squalene with 514.34 mg/L was obtained by the engineered strain SQ-1 grown on YPD-80 medium. Finally, by optimizing the addition concentrations of acetate, citrate and terbinafine, the 731.18 mg/L squalene was produced in the engineered strain SQ-1 with the addition of 0.5 mg/L terbinafine. This work describes the highest reported squalene titer in Y. lipolytica to date. This study will provide the foundation for further engineering Y. lipolytica capable of cost-efficiently producing squalene.

Improved Production of Arachidonic Acid by Combined Pathway Engineering and Synthetic Enzyme Fusion in Yarrowia lipolytica.

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.

Unveiling the Role of Heteroatom Gradient-Distributed Carbon Fibers for Vanadium Redox Flow Batteries with Long Service Life.

The fundamental understanding of electrocatalytic reaction process is anticipated to guide electrode upgradation and acquirement of high-performance vanadium redox flow batteries (VRFBs). Herein, a carbon fiber prototype system with a heteroatom gradient distribution has been developed with enlarged interlayer spacing and a high graphitization that improve the electronic conductivity and accelerate the electrocatalytic reaction, and the mechanism by which gradient-distributed heteroatoms enhance vanadium redox reactions was elucidated with the assistance of density functional theory calculations. All these contributions endow the obtained electrode prominent redox reversibility and durability with only 1.7% decay in energy efficiency over 1000 cycles at 150 mA cm-2 in the VRFBs. Our work sheds light on the significance of elaborated electrode design and impels the in-depth investigation of VRFBs with long service life.

Naringin Activates AMPK Resulting in Altered Expression of SREBPs, PCSK9, and LDLR To Reduce Body Weight in Obese C57BL/6J Mice.

Previous investigations have shown molecular cross-talk among activated adenosine monophosphate-activated protein kinase (AMPK), proprotein convertase subtilisin/kexin type 9 (PCSK9), sterol regulatory element-binding proteins (SREBPs), and low-density lipoprotein receptor (LDLR) and that it may be an innovative pharmacologic objective for treating obesity. We scrutinized the beneficial effect of naringin, a flavanone-7- O-glycoside, on obesity and the mechanisms in the present study. We arbitrarily divided 50 mice into five groups ( n = 10): 25 or 50 or 100 mg/kg/day naringin-treated obese mice (gavage for 8 weeks), untreated obese mice, and C57BL/6J control. After 8 weeks, body weight was 51.8 ± 4.4 in the untreated obese mice group, while the weights were 41.4 ± 4.1, 34.6 ± 2.2, and 28.0 ± 2.3 in 25, 50,100 mg/kg naringin groups, respectively. Moreover, naringin treatment significantly decreased plasma 8-isoprostane (an indicator of the oxidative stress) level, fat weight, liver weight, hepatic total cholesterol concentration, hepatic triglyceride concentration, plasma leptin level, plasma insulin content, plasma low-density lipoprotein cholesterol level, and plasma PCSK9 production concomitantly with down-regulated expression of SREBP-2, PCSK9, and SREBP-1, and up-regulated expression of p-AMPKα and LDLR. The present results suggest that naringin activates AMPK resulting in altered expression of SREBPs, PCSK9, and LDLR to reduce the body weight of obese C57BL/6J mice.

SZSJ protects against insomnia by a decrease in ADMA level and an improvement in DDAH production in sleep-deprived rats.

AIMS: Recent investigations have shown that dimethylarginine dimethylaminohydrolase (DDAH) may be related to sleep disorder. The present study was conducted to test the hypnotic effect of jujubosides from Semen Ziziphi Spinosae (SZSJ) on sleep deprivation and the mechanisms involved. MATERIALS AND METHODS: Forty rats were randomly divided into 4 groups (n = 10): two groups of sleep-deprived rats treated with SZSJ (10 or 30 mg/kg per day), sleep-deprived rats group, and control group. At the end of experiment, cerebral DDAH expression, cerebral asymmetric dimethylarginine (ADMA) level, sleep parameters, behavioral activities, and cerebral neurotransmitters level brain was examined. KEY FINDINGS: Six days after treatment, SZSJ treatment up-regulated cerebral expression of DDAH I and DDAH II concomitantly with elevated parameters of total sleep time, cerebral γ­aminobutyric acid and 5­hydroxytryptamine, and reduced values of forelimb lifting-up frequency and walking time, cerebral 8-isoprostane, ADMA, noradrenaline, and glutamic acid in sleep deprivation of rats. SIGNIFICANCE: The present results suggest that SZSJ Protects against insomnia by a decrease in ADMA level and an improvement in DDAH production in sleep-deprived rats.

Kaempferol modulates Angiopoietin-like protein 2 expression to lessen the mastitis in mice.

BACKGROUND: Mastitis is inflammation of a breast (or udder). Angiopoietin-like protein 2 (ANGPTL2) has been found as a key inflammatory mediator in mastitis. Purpose of this research was to investigate the mechanisms about repressing effect of kaempferol on mastitis. METHODS: Forty mice were randomly divided into 4 groups (n=10): C57BL/6J control mice, untreated murine mastitis, 10mg/kg kaempferol treated murine mastitis (ip), and 30mg/kg kaempferol treated murine mastitis (ip). Primary cultured mouse mammary epithelial cells (MMEC) were indiscriminately divided into seven groups including control group, 10mmol/L vehicle of kaempferol group, 10µmol/L kaempferol treated group, 20µg/mL LPS treated group, 1µmol/L kaempferol plus LPS treated group, 3µmol/L kaempferol plus LPS treated group, and 10µmol/L kaempferol plus LPS treated group. RESULTS: In murine mastitis, kaempferol (10 or 30mg/kg) treatment prevented mastitis development, decreased myeloperoxidase (MPO) production, interleukin (IL)-6 level, tumour necrosis factor-α (TNF-α) concentration, and ANGPTL2 expression. In MMEC, kaempferol (1, 3 or 10µM) reduced MPO production, TNF-α concentration, IL-6 level, and ANGPTL2 expression. CONCLUSIONS: The results in present study show that kaempferol modulates the expression of ANGPTL2 to lessen the mastitis in mice.

Phillyrin lowers body weight in obese mice via the modulation of PPAR/-ANGPTL 4 pathway.

OBJECTIVE: Previous investigations have shown that the peroxisome proliferator activated receptor beta/delta (PPAR/)-angiopoietin-like protein 4 (ANGPTL4) pathways may be a new pharmacologic target for treatment of obesity. The present study was conducted to test the effect of phillyrin, a glucoside, on obesity in mice. METHOD: Fifty mice were randomly divided into 5 groups (n=10): control group (C57BL/6J mice), obese mice group, two groups of obese mice treated with phillyrin (15 or 45mg/kg/day), one group of obese mice treated with PPAR/ agonist GW0742 (3mg/kg/day). Twelve weeks after treatment, body weight, liver weight, fat weight, lipid levels in the liver, serum levels of tumour necrosis factor-(TNF-), leptin, and insulin, expression of PPAR/, ANGPTL4, and AMP-activated protein kinase (AMPK) were determined. RESULTS: Treatment with phillyrin (15 or 45mg/kg) significantly decreased body weight, liver weight, fat weight, hepatic total cholesterol, free fatty acid, and triglyceride concentrations, serum levels of TNF-, leptin, and insulin concomitantly with up-regulated expression of PPAR/, ANGPTL4, and p-AMPK-. In addition, GW0742 has similar effect of phillyrin. CONCLUSIONS: The present results suggest that phillyrin could regulate the PPAR/-ANGPTL 4 pathway to lower body weight in obese C57BL/6J mice.

Elevated Levels of ADMA Are Associated with Lower DDAH2 and Higher PRMT1 in LPS-Induced Endometritis Rats.

Chronic endometritis is a continuous inflammation of uterine endometrium. Recent research has shown that higher asymmetric dimethylarginine (ADMA) levels contribute to endothelial dysfunction. In the present study, we tested whether there is a correlation between endometritis and ADMA in LPS-induced endometritis rat and the mechanisms involved. Thirty-six rats were divided into two groups: blank control group and rat model of endometritis group. The entire infused uterus were removed to observe the changes of histopathology, production of myeloperoxidase (MPO), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, 8-isoprostane, and reactive oxygen species (ROS), and gene expression of dimethylarginine dimethylaminohydrolase 2 (DDAH2), protein-methyl transferase 1 (PRMT1), TNF-α, and IL-6. In endometritis rat group, characteristic histopathologic changes in uteri were observed. The uterine 8-isoprostane, ROS, MPO activity, IL-6 and TNF-α concentrations, PRMT1, IL-6, and TNF-α expressions were significantly elevated, and DDAH2 expression was notably reduced in endometritis group compared with control group. The present findings suggest that elevated levels of ADMA are associated with lower DDAH2 and higher PRMT1 in LPS-induced endometritis rat.

The nonproton ligand of acid-sensing ion channel 3 activates mollusk-specific FaNaC channels via a mechanism independent of the native FMRFamide peptide.

The degenerin/epithelial sodium channel (DEG/ENaC) superfamily of ion channels contains subfamilies with diverse functions that are fundamental to many physiological and pathological processes, ranging from synaptic transmission to epileptogenesis. The absence in mammals of some DEG/ENaCs subfamily orthologues such as FMRFamide peptide-activated sodium channels (FaNaCs), which have been identified only in mollusks, indicates that the various subfamilies diverged early in evolution. We recently reported that the nonproton agonist 2-guanidine-4-methylquinazoline (GMQ) activates acid-sensing ion channels (ASICs), a DEG/ENaC subfamily mainly in mammals, in the absence of acidosis. Here, we show that GMQ also could directly activate the mollusk-specific FaNaCs. Differences in ion selectivity and unitary conductance and effects of substitutions at key residues revealed that GMQ and FMRFamide activate FaNaCs via distinct mechanisms. The presence of two activation mechanisms in the FaNaC subfamily diverging early in the evolution of DEG/ENaCs suggested that dual gating is an ancient feature in this superfamily. Notably, the GMQ-gating mode is still preserved in the mammalian ASIC subfamily, whereas FMRFamide-mediated channel gating was lost during evolution. This implied that GMQ activation may be essential for the functions of mammalian DEG/ENaCs. Our findings provide new insights into the evolution of DEG/ENaCs and may facilitate the discovery and characterization of their endogenous agonists.

Manipulating and detecting the chirpiness of spatial chirp signals via fractional Fourier lenses designed by transformation optics.

Estimating the chirpiness of a spatial chirp signal is important in many optical engineering applications. With the help of transformation optics, a new kind of fractional Fourier transform lens is designed by deforming the conventional graded index lens through conformal mapping, which can manipulate the chirpiness of the input chirp signal. The low-input chirpiness is magnified by the transformation material, and the error of the detection is kept approximately the same; thus, the designed lens has enhanced chirpiness detection precision and distinguishability for low chirpiness. The design is validated by numerical simulations.

Intersubunit physical couplings fostered by the left flipper domain facilitate channel opening of P2X4 receptors.

P2X receptors are ATP-gated trimeric channels with important roles in diverse pathophysiological functions. A detailed understanding of the mechanism underlying the gating process of these receptors is thus fundamentally important and may open new therapeutic avenues. The left flipper (LF) domain of the P2X receptors is a flexible loop structure, and its coordinated motions together with the dorsal fin (DF) domain are crucial for the channel gating of the P2X receptors. However, the mechanism underlying the crucial role of the LF domain in the channel gating remains obscure. Here, we propose that the ATP-induced allosteric changes of the LF domain enable it to foster intersubunit physical couplings among the DF and two lower body domains, which are pivotal for the channel gating of P2X4 receptors. Metadynamics analysis indicated that these newly established intersubunit couplings correlate well with the ATP-bound open state of the receptors. Moreover, weakening or strengthening these physical interactions with engineered intersubunit metal bridges remarkably decreased or increased the open probability of the receptors, respectively. Further disulfide cross-linking and covalent modification confirmed that the intersubunit physical couplings among the DF and two lower body domains fostered by the LF domain at the open state act as an integrated structural element that is stringently required for the channel gating of P2X4 receptors. Our observations provide new mechanistic insights into P2X receptor activation and will stimulate development of new allosteric modulators of P2X receptors.

Icariin improves eNOS/NO pathway to prohibit the atherogenesis of apolipoprotein E-null mice.

Impaired endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) pathway induces atherogenesis. The present study examined whether icariin improves the eNOS/NO pathway to prohibit the atherogenesis of apolipoprotein E-null (ApoE-/-) mice. In vitro, primary human umbilical vein endothelial cells (HUVECs) were randomly divided into 7 groups: control; vehicle; icariin 10; lyphosphatidylcholine (LPC) group; LPC + icariin 1; LPC + icariin 3; and LPC + icariin 10. In vivo, 80 mice were separated randomly into 4 groups (n = 20): control, ApoE-/-, ApoE-/- + icariin 10, and ApoE-/- + icariin 30. ApoE-/- mice had significantly more atherosclerosis in the aortic root together with increased aortic ROS production, body mass, plasma triglyceride (TG) and total cholesterol (TC) concentration, decreased aortic eNOS expression, and plasma NO concentration. LPC (10 µg/mL) treatment induced a big decline in NO level in the conditioned medium and eNOS expression, and an increase in intracellular reactive oxygen species (ROS) production in HUVECs. Icariin treatment decreased atherogenesis, ROS production, body mass, plasma TG concentration, and plasma TC concentration, and increased NO concentration and eNOS expression. These findings suggested icariin could improve eNOS/NO-pathway to prohibit the atherogenesis of apolipoprotein E-null mice by restraining oxidative stress.

[Effects of Rhodopseudomonas palustris PSB06 on Pepper Rhizosphere Microbial Community Structure].

The use of biological pesticide can greatly reduce the soil pollution in the environment. Exploring the effect of biological pesticide on community diversity and distribution of pathogenic bacteria will provide theoretic basis for subsequent researches on biological pesticide micro-ecological control. In order to explore the microbial ecological mechanism of pepper phytophthora blight, this research compared the difference of microbial diversity between rhizosphere soil of infected and healthy plants, and the effects of Rhodopseudomonas palustris PSB06 on microbial diversities of plant rhizosphere soil were investigated using Illumina MiSeq sequencing technology. The results showed that there was less difference in the microbial diversity from the same soil between the seventh day and the fourteenth day. The microbial diversity of rhizosphere soil of healthy plants was higher than that of rhizosphere soil of infected plants. The soil sprayed with Rhodopseudomonas palustris PSB06 exhibited the highest diversity. Moreover, the abundance of Actinomycetes in the rhizosphere soil of healthy plants was higher than that of infected plants, and the highest abundance of Actinomycetes was observed in the soil sprayed with Rhodopseudomonas palustris PSB06. The microbial diversity between rhizosphere soil of infected and healthy plants was significantly different. Spraying Rhodopseudomonas palustris PSB06 could significantly alter the microbial community structure of the soil. It could also increase the diversity of microorganism and the abundance of Actinomycetes in the soil.

Kaempferol inhibits the production of ROS to modulate OPN-αvß3 integrin pathway in HUVECs.

In the present study, we tested the hypothesis that aldosterone regulates osteopontin (OPN)-related signaling pathways to promote nuclear factor κB (NF-κB) activation in primary human umbilical vein endothelial cells (HUVECs) and that kaempferol, a flavonoid compound, blocks those changes. Aldosterone induced productions of reactive oxygen species (ROS), OPN, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) and expression of nicotinamide adenine dinucleotide phosphate-oxidase 4 (Nox4), NF-κB, OPN, alphavbeta3 (αvß3) integrin, and inhibitor of NF-κB alpha phosphorylation (P-IκBα) in HUVEC. HUVECs were pretreated with kaempferol (0, 1, 3, or 10 µM) for 1 h and exposed to aldosterone (10(-6) M) for 24 h. Kaempferol reduced ROS, OPN, NF-κB, IL-6, and TNF-α levels; Nox4, αvß3 integrin; and P-IκBα expressions. The effect of aldosterone was also abrogated by spironolactone (10(-6) M). In addition, vitamin C (20 mmol/L) reduced ROS production. Vitamin C and LM609 (10 µg/mL) treatment decreased expressions of OPN, αvß3 integrin, and NF-κB (P < 0.05 or P < 0.01). The present results suggest that kaempferol may modulate OPN-αvß3 integrin pathway to inhibit NF-κB activation in HUVECs.

Exploration of the Peptide Recognition of an Amiloride-sensitive FMRFamide Peptide-gated Sodium Channel.

FMRFamide (Phe-Met-Arg-Phe-NH2)-activated sodium channel (FaNaC) is an amiloride-sensitive sodium channel activated by endogenous tetrapeptide in invertebrates, and belongs to the epithelial sodium channel/degenerin (ENaC/DEG) superfamily. The ENaC/DEG superfamily differs markedly in its means of activation, such as spontaneously opening or gating by mechanical stimuli or tissue acidosis. Recently, it has been observed that a number of ENaC/DEG channels can be activated by small molecules or peptides, indicating that the ligand-gating may be an important feature of this superfamily. The peptide ligand control of the channel gating might be an ancient ligand-gating feature in this superfamily. Therefore, studying the peptide recognition of FaNaC channels would advance our understanding of the ligand-gating properties of this superfamily of ion channels. Here we demonstrate that Tyr-131, Asn-134, Asp-154, and Ile-160, located in the putative upper finger domain ofHelix aspersaFaNaC (HaFaNaC) channels, are key residues for peptide recognition of this ion channel. Two HaFaNaC specific-insertion motifs among the ENaC/DEG superfamily, residing at the putative α4-α5 linker of the upper thumb domain and the α6-α7 linker of the upper knuckle domain, are also essential for the peptide recognition of HaFaNaC channels. Chemical modifications and double mutant cycle analysis further indicated that those two specific inserts and key residues in the upper finger domain together participate in peptide recognition of HaFaNaC channels. This ligand recognition site is distinct from that of acid-sensing ion channels (ASICs) by a longer distance between the recognition site and the channel gate, carrying useful information about the ligand gating and the evolution of the trimeric ENaC/DEG superfamily of ion channels.

Icariin regulates PRMT/ADMA/DDAH pathway to improve endothelial function.

BACKGROUND: Oxidative stress may affect PRMT/ADMA/DDAH (protein arginine methyltransferases/asymmetric dimethylarginine/dimethylarginine dimethylaminohydrolase) pathway to impair endothelial dysfunction. The present study was carried out to test the effect of icariin on endothelial function and the mechanisms responsible for this. METHODS: Eighty mice at 12 weeks of age were separated randomly into four groups (n = 20): C57BL/6J control, untreated apolipoprotein E-deficient (ApoE(-/-)), two groups of icariin-treated (10 or 30 mg/kg body wt/day, intragastrically) ApoE(-/-). Primary human umbilical vein endothelial cells (HUVECs) were randomly divided into 7 groups: control group, vehicle of icariin (10 µmol/L) group, icariin (10 µmol/L) group, lysophosphatidylcholine (LPC) (10 µg/mL) group, LPC plus icariin (1 µmol/L) group, LPC plus icariin (3 µmol/L) group, and LPC plus icariin (10 µmol/L) group. RESULTS: In ApoE(-/-) mice and primary HUVECs, icariin treatment decreased reactive oxygen species production, PRMT I expression, ADMA level, half-maximum effective concentration of ApoE(-/-) mice aortic rings. Icariin increased DDAH II expression, DDAH activity, maximal relaxation value and endothelium-dependent vasorelaxation in aortic rings from ApoE(-/-) mice (p < 0.05 or p < 0.01). CONCLUSIONS: The present results suggest that icariin regulates PRMT/ADMA/DDAH pathway to improve endothelial function.

Inherent dynamics of head domain correlates with ATP-recognition of P2X4 receptors: insights gained from molecular simulations.

P2X receptors are ATP-gated ion channels involved in many physiological functions, and determination of ATP-recognition (AR) of P2X receptors will promote the development of new therapeutic agents for pain, inflammation, bladder dysfunction and osteoporosis. Recent crystal structures of the zebrafish P2X4 (zfP2X4) receptor reveal a large ATP-binding pocket (ABP) located at the subunit interface of zfP2X4 receptors, which is occupied by a conspicuous cluster of basic residues to recognize triphosphate moiety of ATP. Using the engineered affinity labeling and molecular modeling, at least three sites (S1, S2 and S3) within ABP have been identified that are able to recognize the adenine ring of ATP, implying the existence of at least three distinct AR modes in ABP. The open crystal structure of zfP2X4 confirms one of three AR modes (named AR1), in which the adenine ring of ATP is buried into site S1 while the triphosphate moiety interacts with clustered basic residues. Why architecture of ABP favors AR1 not the other two AR modes still remains unexplored. Here, we examine the potential role of inherent dynamics of head domain, a domain involved in ABP formation, in AR determinant of P2X4 receptors. In silico docking and binding free energy calculation revealed comparable characters of three distinct AR modes. Inherent dynamics of head domain, especially the downward motion favors the preference of ABP for AR1 rather than AR2 and AR3. Along with the downward motion of head domain, the closing movement of loop139-146 and loop169-183, and structural rearrangements of K70, K72, R298 and R143 enabled ABP to discriminate AR1 from other AR modes. Our observations suggest the essential role of head domain dynamics in determining AR of P2X4 receptors, allowing evaluation of new strategies aimed at developing specific blockers/allosteric modulators by preventing the dynamics of head domain associated with both AR and channel activation of P2X4 receptors.