In-silico identification of Tyr232 in AMPKα2 as a dephosphorylation site for the protein tyrosine phosphatase PTP-PEST.

The AMP-activated protein kinase (AMPK) is known to be activated by the protein tyrosine phosphatase non-receptor type 12 (PTP-PEST) under hypoxic conditions. This activation is mediated by tyrosine dephosphorylation of the AMPKα subunit. However, the identity of the phosphotyrosine residues that PTP-PEST dephosphorylates remains unknown. In this study, we first predicted the structure of the complex of the AMPKα2 subunit and PTP-PEST catalytic domain using bioinformatics tools and further confirmed the stability of the complex using molecular dynamics simulations. Evaluation of the protein-protein interfaces indicated that residue Tyr232 is the most likely dephosphorylation site on AMPKα2. In addition, we explored the effect of phosphorylation of PTP-PEST residue Tyr64 on the stability of the complex. Phosphorylation of the highly conserved Tyr64, an interface residue, enhances the stability of the complex via the rearrangement of a network of electrostatic interactions in conjunction with conformational changes in the catalytic WPD loop. We generated a phosphomimetic (PTP-PEST-Y64D) mutant and used co-immunoprecipitation to study the effect of PTP-PEST phosphorylation on AMPKα2 binding. The mutant exhibited an increased affinity for AMPKα2 and corroborated the in-silico predictions. Together, our findings present a plausible structural basis of AMPK regulation by PTP-PEST and show how phosphorylation of PTP-PEST affects its interaction with AMPKα2.

Structure of an iminosugar complex of a glycoside hydrolase family 5 lichenase provides insights into the active site.

TmCel5B is a lichenase belonging to glycoside hydrolase family 5 subfamily 36 (GH5_36). To gain insights into the active site of this subfamily which contains multifunctional endoglycanases, we determined the crystal structure of TmCel5B in complex with an iminosugar, 1-deoxynojiromycin (DNJ). DNJ is bound to the -1 subsite, making a network of non-covalent interactions with the acid/base residue Glu139, the nucleophile Glu259, and with other residues that are conserved across the GH5 family. The catalytic site displayed a Glu-Arg-Glu triad of the catalytic glutamates that is unique to the GH5_36 subfamily. Structural comparison of active sites of GH5_36 homologs revealed divergent residues and loop regions that are likely molecular determinants of homolog-specific properties. Furthermore, a comparative analysis of the binding modes of iminocyclitol complexes of GH5 homologs revealed the structural basis of their binding to GH5 glycosidases, in which the subsite binding location, the interactions of the ligand with specific conserved residues, and the electrostatic interactions of the catalytic glutamates with the ring nitrogen, are crucial.

Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4.

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and ß-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Šresolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure-function relationships within the unique GH4 family.

The protein tyrosine phosphatase PTP-PEST mediates hypoxia-induced endothelial autophagy and angiogenesis via AMPK activation.

Global and endothelial loss of PTP-PEST (also known as PTPN12) is associated with impaired cardiovascular development and embryonic lethality. Although hypoxia is implicated in vascular remodelling and angiogenesis, its effect on PTP-PEST remains unexplored. Here we report that hypoxia (1% oxygen) increases protein levels and catalytic activity of PTP-PEST in primary endothelial cells. Immunoprecipitation followed by mass spectrometry revealed that α subunits of AMPK (α1 and α2, encoded by PRKAA1 and PRKAA2, respectively) interact with PTP-PEST under normoxia but not in hypoxia. Co-immunoprecipitation experiments confirmed this observation and determined that AMPK α subunits interact with the catalytic domain of PTP-PEST. Knockdown of PTP-PEST abrogated hypoxia-mediated tyrosine dephosphorylation and activation of AMPK (Thr172 phosphorylation). Absence of PTP-PEST also blocked hypoxia-induced autophagy (LC3 degradation and puncta formation), which was rescued by the AMPK activator metformin (500 µM). Because endothelial autophagy is a prerequisite for angiogenesis, knockdown of PTP-PEST also attenuated endothelial cell migration and capillary tube formation, with autophagy inducer rapamycin (200 nM) rescuing angiogenesis. In conclusion, this work identifies for the first time that PTP-PEST is a regulator of hypoxia-induced AMPK activation and endothelial autophagy to promote angiogenesis.

A conserved π-helix plays a key role in thermoadaptation of catalysis in the glycoside hydrolase family 4.

Here, we characterize the role of a π-helix in the molecular mechanisms underlying thermoadaptation in the glycoside hydrolase family 4 (GH4). The interspersed π-helix present in a subgroup is evolutionarily related to a conserved α-helix in other orthologs by a single residue insertion/deletion event. The insertional residue, Phe407, in a hyperthermophilic α-glucuronidase, makes specific interactions across the inter-subunit interface. In order to establish the sequence-structure-stability implications of the π-helix, the wild-type and the deletion variant (Δ407) were characterized. The variant showed a significant lowering of melting temperature and optimum temperature for the highest activity. Crystal structures of the proteins show a transformation of the π-helix to a continuous α-helix in the variant, identical to that in orthologs lacking this insertion. Thermodynamic parameters were determined from stability curves representing the temperature dependence of unfolding free energy. Though the proteins display maximum stabilities at similar temperatures, a higher melting temperature in the wild-type is achieved by a combination of higher enthalpy and lower heat capacity of unfolding. Comparisons of the structural changes, and the activity and thermodynamic profiles allow us to infer that specific non-covalent interactions, and the existence of residual structure in the unfolded state, are crucial determinants of its thermostability. These features permit the enzyme to balance the preservation of structure at a higher temperature with the thermodynamic stability required for optimum catalysis.

Evolutionary history of histamine receptors: Early vertebrate origin and expansion of the H3-H4 subtypes.

Histamine receptors belonging to the superfamily of G protein-coupled receptors (GPCRs) mediate the diverse biological effects of biogenic histamine. They are classified into four phylogenetically distinct subtypes H1-H4, each with a different binding affinity for histamine and divergent downstream signaling pathways. Here we present the evolutionary history of the histamine receptors using a phylogenetic approach complemented with comparative genomics analyses of the sequences, gene structures, and synteny of gene neighborhoods. The data indicate the earliest emergence of histamine-mediated GPCR signaling by a H2 in a prebilaterian ancestor. The analyses support a revised classification of the vertebrate H3-H4 receptor subtypes. We demonstrate the presence of the H4 across vertebrates, contradicting the currently held notion that H4 is restricted to mammals. These non-mammalian vertebrate H4 orthologs have been mistaken for H3. We also identify the presence of a new H3 subtype (H3B), distinct from the canonical H3 (H3A), and propose that the H3A, H3B, and H4 likely emerged from a H3 progenitor through the 1R/2R whole genome duplications in an ancestor of the vertebrates. It is apparent that the ability of the H1, H2, and H3-4 to bind histamine was acquired convergently. We identified genomic signatures suggesting that the H1 and H3-H4 shared a last common ancestor with the muscarinic receptor in a bilaterian predecessor whereas, the H2 and the α-adrenoreceptor shared a progenitor in a prebilaterian ancestor. Furthermore, site-specific analysis of the vertebrate subtypes revealed potential residues that may account for the functional divergence between them.

Crystallographic Snapshots of the Dunathan and Quinonoid Intermediates provide Insights into the Reaction Mechanism of Group II Decarboxylases.

PLP-dependent enzymes catalyze a plethora of chemical reactions affecting diverse physiological functions. Here we report the structural determinants of the reaction mechanism in a Group II PLP-dependent decarboxylase by assigning two early intermediates. The in-crystallo complexes of the PLP bound form, and the Dunathan and quinonoid intermediates, allowed direct observation of the active site interactions. The structures reveal that a subtle rearrangement of a conserved Arg residue in concert with a water-mediated interaction with the carboxylate of the Dunathan intermediate, appears to directly stabilize the alignment and facilitate the release of CO2 to yield the quinonoid. Modeling indicates that the conformational change of a dynamic catalytic loop to a closed form controls a conserved network of hydrogen bond interactions between catalytic residues to protonate the quinonoid. Our results provide a structural framework to elucidate mechanistic roles of residues that govern reaction specificity and catalysis in PLP-dependent decarboxylation.

Structural insights into the mechanism of internal aldimine formation and catalytic loop dynamics in an archaeal Group II decarboxylase.

Formation of the internal aldimine (LLP) is the first regulatory step that activates pyridoxal 5'-phosphate (PLP) dependent enzymes. The process involves a nucleophilic attack on PLP by an active site Lys residue, followed by proton transfers resulting in a carbinolamine (CBA) intermediate that undergoes dehydration to form the aldimine. Despite a general understanding of the pathway, the structural basis of the mechanistic roles of specific residues in each of these steps is unclear. Here we determined the crystal structure of the LLP form (holo-form) of a Group II PLP-dependent decarboxylase from Methanocaldococcus jannaschii (MjDC) at 1.7 Šresolution. By comparing the crystal structure of MjDC in the LLP form with that of the pyridoxal-P (non-covalently bound aldehyde) form, we demonstrate structural evidence for a water-mediated mechanism of LLP formation. A conserved extended hydrogen-bonding network around PLP coupled to the pyridinyl nitrogen influences activation and catalysis by affecting the electronic configuration of PLP. Furthermore, the two cofactor bound forms revealed open and closed conformations of the catalytic loop (CL) in the absence of a ligand, supporting a hypothesis for a regulatory link between LLP formation and CL dynamics. The evidence suggests that activation of Group II decarboxylases involves a complex interplay of interactions between the electronic states of PLP, the active site micro-environment and CL dynamics.

Structure of an α-glucuronidase in complex with Co2+ and citrate provides insights into the mechanism and substrate recognition in the family 4 glycosyl hydrolases.

Glycosyl hydrolases belonging to the family 4 (GH4) use a unique redox-based NAD+-dependent reaction mechanism involving anionic intermediates and requires a divalent metal ion and reducing conditions for catalytic activity. These enzymes display wide specificity and selectivity for their substrates. However, the structural basis of substrate binding, recognition and specificity remains poorly studied. Here, we report the crystal structure of Thermotoga maritima TmAgu4B, a GH4 α-glucuronidase, in complex with Co2+ and citrate. Analysis of GH4 structures show that the metal ion is present in a conserved octahedral coordination with conserved side chain atoms, the ligand atoms and an invariant water molecule. The data provides the first structural evidence for a metal-activated hydroxide ion that acts as the general base to deprotonate the C3-hydroxyl group of the glycone, a rate-limiting step in the mechanism. Furthermore, the citrate binding mode in the active site is analogous to a bound glucuronide substrate and provides insights into the mode of substrate interaction with the metal ion, the active site residues and, the structural basis of substrate recognition in a GH4 α-glucuronidase.

Amarogentin, a secoiridoid glycoside, activates AMP- activated protein kinase (AMPK) to exert beneficial vasculo-metabolic effects.

INTRODUCTION: AMP-activated protein kinase (AMPK) is a drug target for treatment of metabolic and cardiovascular complications. Extracts of Gentianaceace plants exhibit anti-diabetic and anti-atherosclerotic effects, however, whether their phyto-constitutents activate AMPK remains to be determined. METHODS: Molecular docking of Gentiana lutea constituents was performed with crystal structure of human α2ß1γ1 trimeric AMPK (PDB ID: 4CFE). Binding of Amarogentin (AG) to α2 subunit was confirmed through isothermal titration calorimetry (ITC) and in vitro kinase assays were performed. L6 myotube, HUH7 and endothelial cell cultures were employed to validate in silico and in vitro observations. Lipid lowering and anti-atherosclerotic effects were confirmed in streptozotocin induced diabetic mice via biochemical measurements and through heamatoxylin and eosin, Masson's trichrome and Oil Red O staining. RESULTS: AG interacts with the α2 subunit of AMPK and activates the trimeric kinase with an EC50 value of 277 pM. In cell culture experiments, AG induced phosphorylation of AMPK as well as its downstream targets, acetyl-coA-carboxylase (ACC) and endothelial nitric oxide synthase (eNOS). Additionally, it enhanced glucose uptake in myotubes and blocked TNF-α induced endothelial inflammation. Oral supplementation of AG significantly attenuated diabetes-mediated neointimal thickening, and collagen and lipid deposition in the aorta. It also improved circulating levels of lipids and liver function in diabetic mice. CONCLUSION: In conclusion, AG exerts beneficial vasculo-metabolic effects by activating AMPK. GENERAL SIGNIFICANCE: Amarogentin, a naturally occurring secoiridoid glycoside, is a promising lead for design and synthesis of novel drugs for treatment and management of dyslipidemia and cardiovascular diseases.

Molecular evolution of proteins mediating mitochondrial fission-fusion dynamics.

Eukaryotes employ a subset of dynamins to mediate mitochondrial fusion and fission dynamics. Here we report the molecular evolution and diversification of the dynamin-related mitochondrial proteins that drive the fission (Drp1) and the fusion processes (mitofusin and OPA1). We demonstrate that the three paralogs emerged concurrently in an early mitochondriate eukaryotic ancestor. Furthermore, multiple independent duplication events from an ancestral bifunctional fission protein gave rise to specialized fission proteins. The evolutionary history of these proteins is marked by transformations that include independent gain and loss events occurring at the levels of entire genes, specific functional domains, and intronic regions. The domain level variations primarily comprise loss-gain of lineage specific domains that are present in the terminal regions of the sequences.

Crystal structure of yeast xylose reductase in complex with a novel NADP-DTT adduct provides insights into substrate recognition and catalysis.

Aldose reductases (ARs) belonging to the aldo-keto reductase (AKR) superfamily catalyze the conversion of carbonyl substrates into their respective alcohols. Here we report the crystal structures of the yeast Debaryomyces nepalensis xylose reductase (DnXR, AKR2B10) in the apo form and as a ternary complex with a novel NADP-DTT adduct. Xylose reductase, a key enzyme in the conversion of xylose to xylitol, has several industrial applications. The enzyme displayed the highest catalytic efficiency for l-threose (138 ± 7 mm-1 ·s-1 ) followed by d-erythrose (30 ± 3 mm-1 ·s-1 ). The crystal structure of the complex reveals a covalent linkage between the C4N atom of the nicotinamide ring of the cosubstrate and the S1 sulfur atom of DTT and provides the first structural evidence for a protein mediated NADP-low-molecular-mass thiol adduct. We hypothesize that the formation of the adduct is facilitated by an in-crystallo Michael addition of the DTT thiolate to the specific conformation of bound NADPH in the active site of DnXR. The interactions between DTT, a four-carbon sugar alcohol analog, and the enzyme are representative of a near-cognate product ternary complex and provide significant insights into the structural basis of aldose binding and specificity and the catalytic mechanism of ARs. DATABASE: Structural data are available in the PDB under the accession numbers 5ZCI and 5ZCM.

Role of an N-terminal extension in stability and catalytic activity of a hyperthermostable α/ß hydrolase fold esterase.

The carbohydrate esterase family 7 (CE7) enzymes catalyze the deacetylation of acetyl esters of a broad range of alcohols and is unique in its activity towards cephalosporin C. The CE7 fold contains a conserved N-terminal extension that distinguishes it from the canonical α/ß hydrolase fold. The hexameric quaternary structure indicates that the N-terminus may affect activity and specificity by controlling access of substrates to the buried active sites via an entrance tunnel. In this context, we characterized the catalytic parameters, conformation and thermal stability of two truncation variants lacking four and ten residues of the N-terminal region of the hyperthermostable Thermotoga maritima CE7 acetyl esterase (TmAcE). The truncations did not affect the secondary structure or the fold but modulated the oligomerization dynamics. A modest increase was observed in substrate specificity for acetylated xylose compared with acetylated glucose. A drastic reduction of ~30-40°C in the optimum temperature for activity of the variants indicated lower thermal stability. The loss of hyperthermostability appears to be an indirect effect associated with an increase in the conformational flexibility of an otherwise rigid neighboring loop containing a catalytic triad residue. The results suggest that the N-terminal extension was evolutionarily selected to preserve the stability of the enzyme.

A temperature-responsive gene in sorghum encodes a glycine-rich protein that interacts with calmodulin.

Imposition of different biotic and abiotic stress conditions results in an increase in intracellular levels of Ca2+ which is sensed by various sensor proteins. Calmodulin (CaM) is one of the best studied transducers of Ca2+ signals. CaM undergoes conformational changes upon binding to Ca2+ and interacts with different types of proteins, thereby, regulating their activities. The present study reports the cloning and characterization of a sorghum cDNA encoding a protein (SbGRBP) that shows homology to glycine-rich RNA-binding proteins. The expression of SbGRBP in the sorghum seedlings is modulated by heat stress. The SbGRBP protein is localized in the nucleus as well as in cytosol, and shows interaction with CaM that requires the presence of Ca2+. SbGRBP depicts binding to single- and also double-stranded DNA. Fluorescence spectroscopic analyses suggest that interaction of SbGRBP with nucleic acids may be modulated after binding with CaM. To our knowledge, this is the first study to provide evidence for interaction of a stress regulated glycine-rich RNA-binding protein with CaM.

Structural role of a conserved active site cis proline in the Thermotoga maritima acetyl esterase from the carbohydrate esterase family 7.

A conserved cis proline residue located in the active site of Thermotoga maritima acetyl esterase (TmAcE) from the carbohydrate esterase family 7 (CE7) has been substituted by alanine. The residue was known to play a crucial role in determining the catalytic properties of the enzyme. To elucidate the structural role of the residue, the crystal structure of the Pro228Ala variant (TmAcEP228A ) was determined at 2.1 Å resolution. The replacement does not affect the overall secondary, tertiary, and quaternary structures and moderately decreases the thermal stability. However, the wild type cis conformation of the 227-228 peptide bond adopts a trans conformation in the variant. Other conformational changes in the tertiary structure are restricted to residues 222-226, preceding this peptide bond and are located away from the active site. Overall, the results suggest that the conserved proline residue is responsible for the cis conformation of the peptide and shapes the geometry of the active site. Elimination of the pyrrolidine ring results in the loss of van der Waals and hydrophobic interactions with both the alcohol and acyl moeities of the ester substrate, leading to significant impairment of the activity and perturbation of substrate specificity. Furthermore, a cis-to-trans conformational change arising out of residue changes at this position may be associated with the evolution of divergent activity, specificity, and stability properties of members constituting the CE7 family. Proteins 2017; 85:694-708. © 2016 Wiley Periodicals, Inc.

Phosphatidylserine and Phosphatidylethanolamine Bind to Protein Z Cooperatively and with Equal Affinity.

Protein Z (PZ) is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI) and accelerates the rate of ZPI-mediated inhibition of factor Xa (fXa) by more than 1000-fold in the presence of Ca2+ and phospholipids. PZ promotion of the ZPI-fXa interaction results from the anchoring of the Gla domain of PZ onto phospholipid surfaces and positioning the bound ZPI in close proximity to the Gla-anchored fXa, forming a ternary complex of PZ/ZPI/fXa. Although interaction of PZ with phospholipid membrane appears to be absolutely crucial for its cofactor activity, little is known about the binding of different phospholipids to PZ. The present study was conceived to understand the interaction of different phospholipids with PZ. Experiments with both soluble lipids and model membranes revealed that PZ binds to phosphatidylserine (PS) and phosphatidylethanolamine (PE) with equal affinity (Kd~48 µM); further, PS and PE bound to PZ synergistically. Equilibrium dialysis experiments revealed two lipid-binding sites for both PS and PE. PZ binds with weaker affinity to other phospholipids, e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylcholine and binding of these lipids is not synergistic with respect to PS. Both PS and PE -containing membranes supported the formation of a fXa-PZ complex. PZ protection of fXa from antithrombin inhibition were also shown to be comparable in presence of both PS: PC and PE: PC membranes. These findings are particularly important and intriguing since they suggest a special affinity of PZ, in vivo, towards activated platelets, the primary membrane involved in blood coagulation process.

Crystal structure of Thermotoga maritima acetyl esterase complex with a substrate analog: Insights into the distinctive substrate specificity in the CE7 carbohydrate esterase family.

The carbohydrate esterase family 7 (CE7) members are acetyl esterases that possess unusual substrate specificity for cephalosporin C and 7-amino-cephalosporanic acid. This family containing the α/ß hydrolase fold has a distinctive substrate profile that allows it to carry out hydrolysis of esters containing diverse alcohol moieties while maintaining narrow specificity for an acetate ester. Here we investigate the structural basis of this preference for small acyl groups using the crystal structure of the thermostable Thermotoga maritima CE7 acetyl esterase (TmAcE) complexed with a non-cognate substrate analog. The structure determined at 1.86 Å resolution provides direct evidence for the location of the largely hydrophobic and rigid substrate binding pocket in this family. Furthermore, a three-helix insertion domain near the catalytic machinery shapes the substrate binding site. The structure reveals two residues (Pro228 and Ile276) which constitute a hydrophobic rigid binding surface for the acyl group of the ester and thus restricts the size of the acyl group that be accommodated. In combination with previous literature on kinetic properties of the enzyme, our studies suggest that these residues determine the unique specificity of the TmAcE for short straight chain esters. The structure provides a template for focused attempts to engineer the CE7 enzymes for enhanced stability, selectivity or activity for biocatalytic applications.

An extended loop in CE7 carbohydrate esterase family is dispensable for oligomerization but required for activity and thermostability.

The carbohydrate esterase family 7 (CE7) belonging to the α/ß hydrolase superfamily contains a structurally conserved loop extension element relative to the canonical α/ß hydrolase fold. This element called the ß-interface loop contributes 20-30% of the total buried surface area at intersubunit interfaces of the functional hexameric state. To test whether this loop is an enabling region for the structure and function of the oligomeric assembly, we designed a truncation variant of the thermostable CE7 acetyl esterase from Thermotoga maritima (TmAcE). Although deletion of 26 out of 40 residues in the loop had little impact on the hexamer formation, the variant exhibited altered dynamics of the oligomeric assembly and a loss of thermal stability. Furthermore, the mutant lacked catalytic activity. Crystal structures of the variant and a new crystal form of the wild type protein determined at 2.75Å and 1.76Å, respectively, provide a rationale for the properties of the variant. The hexameric assembly in the variant is identical to that of the wild type and differed only in the lack of buried surface area interactions at the original intersubunit interfaces. This is accompanied by disorder in an extended region of the truncated loop that consequently induces disorder in the neighboring oxyanion hole loop. Overall, the results suggest that the ß-interface loop in CE7 enzymes is dispensable for the oligomeric assembly. Rather, the loop extension event was evolutionarily selected to regulate activity, conformational flexibility and thermal stability.

The GPCR repertoire in the demosponge Amphimedon queenslandica: insights into the GPCR system at the early divergence of animals.

BACKGROUND: G protein-coupled receptors (GPCRs) play a central role in eukaryotic signal transduction. However, the GPCR component of this signalling system, at the early origins of metazoans is not fully understood. Here we aim to identify and classify GPCRs in Amphimedon queenslandica (sponge), a member of an earliest diverging metazoan lineage (Porifera). Furthermore, phylogenetic comparisons of sponge GPCRs with eumetazoan and bilaterian GPCRs will be essential to our understanding of the GPCR system at the roots of metazoan evolution. RESULTS: We present a curated list of 220 GPCRs in the sponge genome after excluding incomplete sequences and false positives from our initial dataset of 282 predicted GPCR sequences obtained using Pfam search. Phylogenetic analysis reveals that the sponge genome contains members belonging to four of the five major GRAFS families including Glutamate (33), Rhodopsin (126), Adhesion (40) and Frizzled (3). Interestingly, the sponge Rhodopsin family sequences lack orthologous relationships with those found in eumetazoan and bilaterian lineages, since they clustered separately to form sponge specific groups in the phylogenetic analysis. This suggests that sponge Rhodopsins diverged considerably from that found in other basal metazoans. A few sponge Adhesions clustered basal to Adhesion subfamilies commonly found in most vertebrates, suggesting some Adhesion subfamilies may have diverged prior to the emergence of Bilateria. Furthermore, at least eight of the sponge Adhesion members have a hormone binding motif (HRM domain) in their N-termini, although hormones have yet to be identified in sponges. We also phylogenetically clarified that sponge has homologs of metabotropic glutamate (mGluRs) and GABA receptors. CONCLUSION: Our phylogenetic comparisons of sponge GPCRs with other metazoan genomes suggest that sponge contains a significantly diversified set of GPCRs. This is evident at the family/subfamily level comparisons for most GPCR families, in particular for the Rhodopsin family of GPCRs. In summary, this study provides a framework to perform future experimental and comparative studies to further verify and understand the roles of GPCRs that predates the divergence of bilaterian and eumetazoan lineages.

Evolutionary history of the neuropeptide S receptor/neuropeptide S system.

The neuropeptide S receptor (NPSR) belongs to the G protein-coupled receptor (GPCR) superfamily and is activated by the neuropeptide S (NPS). Although recently discovered, the vertebrate NPSR-NPS system has been established as an important signaling system in the central nervous system and is involved in physiological processes such as locomotor activity, wakefulness, asthma pathogenesis, anxiety and food intake. The availability of a large number of genome sequences from multiple bilaterian lineages has provided an opportunity to establish the evolutionary history of the system. This review describes the origin and the molecular evolution of the NPSR-NPS system using data derived primarily from comparative genomic analyses. These analyses indicate that the NPSR-NPS system and the vasopressin-like receptor-vasopressin/oxytocin peptide (VPR-VP/OT) system originated from a single system in an ancestral bilaterian. Multiple duplications of this ancestral system gave rise to the bilaterian VPR-VP/OT system and to the protostomian cardioacceleratory peptide receptor-cardioacceleratory peptide (CCAPR-CCAP) system and to the NPSR-NPS system in the deuterostomes. Gene structure features of the receptors were consistent with the orthology annotations derived from phylogenetic analyses. The orthology of the peptide precursors closely paralleled that of the receptors suggesting an ancient coevolution of the receptor-peptide pair. An important challenge for the coevolution hypothesis will be to establish the molecular and structural basis of the divergence between orthologous receptor-ligand pairs in this system.