Site-specific unglycosylation to improve crystallization of the metabotropic glutamate receptor 3 extracellular domain.

Metabotropic glutamate receptors (mGluRs) are involved in the regulation of many physiological and pathological processes in the central nervous system. The extracellular domain (ECD) of mGluR subtype 3 (mGluR3) was produced using the baculovirus expression system and purified from the culture medium. However, the recombinant protein showed heterogeneity in molecular weight on SDS-PAGE analysis. It was found that the unglycosylation of Asn414 significantly reduced the heterogeneity. Consequently, three site-specifically unglycosylated mutant proteins of mGluR3 ECD, replacing Asn414 only or replacing Asn414 in combination with other glycosylation sites, were successfully crystallized in the presence of L-glutamate. Among them, crystals of the N414/439Q mutant diffracted X-rays to 2.35 A resolution using synchrotron radiation. The crystal belonged to the monoclinic space group P2(1), with unit-cell parameters a = 84.0, b = 97.5, c = 108.1 A, beta = 93.0 degrees . Assuming the presence of two protomers per crystallographic asymmetric unit, the Matthews coefficient V(M) was calculated to be 3.5 A(3) Da(-1) and the solvent content was 65%.

Structural insight into PPARgamma activation through covalent modification with endogenous fatty acids.

Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor that regulates lipid homeostasis, and several fatty acid metabolites have been identified as PPARgamma ligands. Here, we present four crystal structures of the PPARgamma ligand binding domain (LBD) covalently bound to endogenous fatty acids via a unique cysteine, which is reportedly critical for receptor activation. The structure analyses of the LBD complexed with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) revealed that the covalent binding of 15d-PGJ(2) induced conformational changes in the loop region following helix H2', and rearrangements of the side-chain network around the created covalent bond in the LBD. Point mutations of these repositioned residues on the loop and helix H3 almost completely abolished PPARgamma activation by 15d-PGJ(2), indicating that the observed structural alteration may be crucial for PPARgamma activation by the endogenous fatty acid. To address the issue of partial agonism of endogenous PPARgamma ligands, we took advantage of a series of oxidized eicosatetraenoic acids (oxoETEs) as covalently bound ligands to PPARgamma. Despite similar structural and chemical properties, these fatty acids exhibited distinct degrees of transcriptional activity. Crystallographic studies, using two of the oxoETE/PPARgamma LBD complexes, revealed that transcriptional strength of each oxoETE is associated with the difference in the loop conformation, rather than the interaction between each ligand and helix H12. These results suggest that the loop conformation may be responsible for the modulation of PPARgamma activity. Based on these results, we identified novel agonists covalently bound to PPARgamma by in silico screening and a cell-based assay. Our crystallographic study of LBD complexed with nitro-233 demonstrated that the expected covalent bond is indeed formed between this newly identified agonist and the cysteine. This study presents the structural basis for the activation and modulation mechanism of PPARgamma through covalent modification with endogenous fatty acids.

Direct detection of the interaction between recombinant soluble extracellular regions in the heterodimeric metabotropic gamma-aminobutyric acid receptor.

The gamma-aminobutyric acid, type B (GABAB) receptor is a heterodimeric receptor consisting of two complementary subunits, GABAB1 receptor (GBR1) and GABAB2 receptor (GBR2). GBR1 is responsible for GABA binding, whereas GBR2 is considered to perform a critical role in signal transduction toward downstream targets. Therefore, precise communication between GBR1 and GBR2 is thought to be essential for the proper signal transduction process. However, biochemical data describing the interaction of the two subunits, especially for the extracellular regions, are not sufficient. Thus we began by developing a protein expression system of the soluble extracellular regions. One of the soluble recombinant GBR1 proteins exhibited a ligand binding ability, which is similar to that of the full-length GBR1, and thus the ligand-binding domain was determined. Direct interaction between GBR1 and GBR2 extracellular soluble fragments was confirmed by co-expression followed by affinity column chromatography and a sucrose density gradient sedimentation. In addition, we also found homo-oligomeric states of these soluble extracellular regions. The interaction between the two soluble extracellular regions caused the enhancement of the agonist affinity for GBR1 as previously reported in a cell-based assay. These results not only open the way to future structural studies but also highlight the role of the interaction between the extracellular regions, which controls agonist affinity to the heterodimeric receptor.

Structural insights into the PIP2 recognition by syntenin-1 PDZ domain.

Lipid-mediated regulatory mechanism of the C-terminal ligand binding to PDZ domains is not fully understood, despite their roles in subcellular organization. Here, we provide structural insights into the phosphatidylinositol 4,5-bisphosphate (PIP(2)) recognition mode of a PDZ domain, as revealed from the crystal structure of the phosphate-bound PDZ domain. Two adjacent phosphate ions bind to the basic residues close to the amino terminus of the alpha2 helix in the Tamalin PDZ domain, reflecting an interaction mode of the two phosphate groups of PIP(2). Based on the observed location of the two phosphate molecules within the PDZ domain, we built the docking model of PIP(2) with the PDZ domain of the well-known PIP(2)-binding protein, syntenin-1. This model suggests that the hydrophobic diacylglycerol group of PIP(2) could contact the ligand-binding groove of the PDZ domain. These structural features well explain biological phenomena, which were previously reported for the PIP(2)-mediated PDZ ligand-binding regulation.

Expression, purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of metabotropic glutamate receptor 7.

Glutamate is the major excitatory neurotransmitter and its metabotropic glutamate receptor (mGluR) plays an important role in the central nervous system. The ligand-binding domain (LBD) of mGluR subtype 7 (mGluR7) was produced using the baculovirus expression system and purified from the culture medium. The purified protein was characterized by gel-filtration chromatography, SDS-PAGE and a ligand-binding assay. Crystals of mGluR7 LBD were grown at 293 K by the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 3.30 A resolution using synchrotron radiation and belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 92.4, c = 114.3 A. Assuming the presence of one protomer per crystallographic asymmetric unit, the Matthews coefficient V(M) was calculated to be 2.5 A3 Da(-1) and the solvent content was 51%.

Structures of the extracellular regions of the group II/III metabotropic glutamate receptors.

Metabotropic glutamate receptors play major roles in the activation of excitatory synapses in the central nerve system. We determined the crystal structure of the entire extracellular region of the group II receptor and that of the ligand-binding region of the group III receptor. A comparison among groups I, II, and III provides the structural basis that could account for the discrimination of group-specific agonists. Furthermore, the structure of group II includes the cysteine-rich domain, which is tightly linked to the ligand-binding domain by a disulfide bridge, suggesting a potential role in transmitting a ligand-induced conformational change into the downstream transmembrane region. The structure also reveals the lateral interaction between the two cysteine-rich domains, which could stimulate clustering of the dimeric receptors on the cell surface. We propose a general activation mechanism of the dimeric receptor coupled with both ligand-binding and interprotomer rearrangements.

Crystal structures of autoinhibitory PDZ domain of Tamalin: implications for metabotropic glutamate receptor trafficking regulation.

Metabotropic glutamate receptors (mGluRs) function as neuronal G-protein-coupled receptors and this requires efficient membrane targeting through associations with cytoplasmic proteins. However, the molecular mechanism regulating mGluR cell-surface trafficking remains unknown. We report here that mGluR trafficking is controlled by the autoregulatory assembly of a scaffold protein Tamalin. In the absence of mGluR, Tamalin self-assembles into autoinhibited conformations, through its PDZ domain and C-terminal intrinsic ligand motif. X-ray crystallographic analyses visualized integral parts of the oligomeric self-assemblies of Tamalin, which require not only the novel hydrophobic dimerization interface but also canonical and noncanonical PDZ/ligand autoinhibitory interactions. The mGluR cytoplasmic region can competitively bind to Tamalin at a higher concentration, disrupting weak inhibitory interactions. The atomic view of mGluR association suggests that this rearrangement is dominated by electrostatic attraction and repulsion. We also observed in mammalian cells that the association liberates the intrinsic ligand toward a motor protein receptor, thereby facilitating mGluR cell-surface trafficking. Our study suggests a novel regulatory mechanism of the PDZ domain, by which Tamalin switches between the trafficking-inhibited and -active forms depending on mGluR association.

The WD40-repeat protein Pwp1p associates in vivo with 25S ribosomal chromatin in a histone H4 tail-dependent manner.

The tails of core histones (H2A, H2B, H3 and H4) are critical for the regulation of chromatin dynamics. Each core histone tail is specifically recognized by various tail binding proteins. Here we screened for budding yeast histone H4-tail binding proteins in a protein differential display approach by two-dimensional gel electrophoresis (2DGE). To obtain highly enriched chromatin proteins, we used a Mg2+-dependent chromatin oligomerization technique. The Mg2+-dependent oligomerized chromatin from H4-tail deleted cells was compared with that from wild-type cells. We used mass spectrometry to identify 22 candidate proteins whose amounts were reduced in the oligomerized chromatin from the H4-tail deleted cells. A Saccharomyces Genome Database search revealed 10 protein complexes, each of which contained more than two candidate proteins. Interestingly, 7 out of the 10 complexes have the potential to associate with the H4-tail. We obtained in vivo evidence, by a chromatin immunoprecipitation assay, that one of the candidate proteins, Pwp1p, associates with the 25S ribosomal DNA (rDNA) chromatin in an H4-tail-dependent manner. We propose that the complex containing Pwp1p regulates the transcription of rDNA. Our results demonstrate that the protein differential display approach by 2DGE, using a histone-tail mutant, is a powerful method to identify histone-tail binding proteins.

The region of CQQQKPQRRP of PGC-1alpha interacts with the DNA-binding complex of FXR/RXRalpha.

PGC-1alpha co-activates transcription by several nuclear receptors. To study the interaction among PGC-1alpha, RXRalpha/FXR, and DNA, we performed electrophoresis mobility shift assays. The RXRalpha/FXR proteins specifically bound to DNA containing the IR-1 sequence in the absence of ligand. When the fusion protein of GST-PGC-1alpha was added to the mixture of RXRalpha/FXR/DNA, the ligand-influenced retardation of the mobility was observed. The ligand for RXRalpha (9-cis-retinoic acid) was necessary for this retardation, whereas, the ligand for FXR, chenodeoxycholic acid, barely had an effect. The results obtained using truncated PGC-1alpha proteins suggested that two regions are necessary for PGC-1alpha to interact with the DNA-binding complex of RXRalpha/FXR. One is the region of the second leucine-rich motif, and the other is that of the amino acid sequence CQQQKPQRRP, present between the second and third leucine-rich motifs. The results obtained with the SPQSS mutation for KPQRR suggested that the basic amino acids are important for the interaction.

Spectroscopic analyses of the binding kinetics of 15d-PGJ2 to the PPARgamma ligand-binding domain by multi-wavelength global fitting.

PPARgamma (peroxisome proliferator-activated receptor gamma) is a nuclear receptor that is activated by natural lipid metabolites, including 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2). We previously reported that several oxidized lipid metabolites covalently bind to PPARgamma through a Michael-addition to activate transcription. To separate the ligand-entering (dock) and covalent-binding (lock) steps in PPARgamma activation, we investigated the binding kinetics of 15d-PGJ2 to the PPARgamma LBD (ligand-binding domain) by stopped-flow spectroscopy. We analysed the spectral changes of 15d-PGJ2 by multi-wavelength global fitting based on a two-step chemical reaction model, in which an intermediate state represents the 15d-PGJ2-PPARgamma complex without covalent binding. The extracted spectrum of the intermediate state in wild-type PPARgamma was quite similar to the observed spectrum of 15d-PGJ2 in the C285S mutant, which cannot be activated by 15d-PGJ2, indicating that the complex remains in the inactive, intermediate state in the mutant. Thus 'lock' rather than 'dock' is one of the critical steps in PPARgamma activation by 15d-PGJ2.

Alpha,beta-unsaturated ketone is a core moiety of natural ligands for covalent binding to peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma (PPARgamma) functions in various biological processes, including macrophage and adipocyte differentiation. Several natural lipid metabolites have been shown to activate PPARgamma. Here, we report that some PPARgamma ligands, including 15-deoxy-Delta12,14-prostaglandin J2, covalently bind to a cysteine residue in the PPARgamma ligand binding pocket through a Michael addition reaction by an alpha,beta-unsaturated ketone. Using rhodamine-maleimide as well as mass spectroscopy, we showed that the binding of these ligands is covalent and irreversible. Consistently, mutation at the cysteine residue abolished abilities of these ligands to activate PPARgamma, but not of BRL49653, a non-covalent synthetic agonist, indicating that covalent binding of the alpha,beta-unsaturated ketone in the natural ligands was required for their transcriptional activities. Screening of lipid metabolites containing the alpha,beta-unsaturated ketone revealed that several other oxidized metabolites of hydroxyeicosatetraenoic acid, hydroxyeicosadecaenoic acid, and prostaglandins can also function as novel covalent ligands for PPARgamma. We propose that PPARgamma senses oxidation of fatty acids by recognizing such an alpha,beta-unsaturated ketone as a common moiety.

Rational discovery of a novel interface for a coactivator in the peroxisome proliferator-activated receptor gamma: theoretical implications of impairment in type 2 diabetes mellitus.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is important to adipocyte differentiation and glucose homeostasis, and mutations in the gene have been observed in type 2 diabetes mellitus. The mutated residues, V290 and P467, bind to neither ligands nor a coactivator peptide in the reported crystal structures of the PPARgamma ligand binding domain. To understand the mechanism of type 2 diabetes mellitus caused by germline mutations in the PPARgamma ligand-binding domain, theoretical models of the PPARgamma-ligand-coactivator complex were built at an atomic resolution. In the models, the secondary coactivator peptide was docked next to the conventional coactivator peptide, which both contain the LXXLL motif. The secondary interface in PPARgamma for the secondary coactivator peptide has not been demonstrated by experiments. Binding energy calculations of the complex, considering the solvent effect, revealed that the secondary coactivator peptide, derived from nuclear receptor box 1 of steroid receptor coactivator 1, can be favorably bound to the secondary interface. The secondary coactivator peptide forms hydrogen bonds and a hydrophobic core with PPARgamma and the primary coactivator peptide. Next, we applied mutations to PPARgamma in silico and found that the V290M mutation, observed in type 2 diabetes mellitus, adversely affected the binding of the secondary peptide. Thus, our model provides structural insight into the impairment of PPARgamma function in type 2 diabetes mellitus.

Phosphorylation and recruitment of Syk by immunoreceptor tyrosine-based activation motif-based phosphorylation of tamalin.

Tamalin is a scaffold protein that forms a multiple protein assembly including metabotropic glutamate receptors (mGluRs) and several postsynaptic and protein-trafficking scaffold proteins in distinct mode of protein-protein association. In the present investigation, we report that tamalin possesses a typical immunoreceptor tyrosine-based activation motif (ITAM), which enables Syk kinase to be recruited and phosphorylated by the Src family kinases. Coimmunoprecipitation analysis of rat brain membrane fractions showed that tamalin is present in a multimolecular protein assembly comprising not only mGluR1 but also c-Src, Fyn, and a protein phosphatase, SHP-2. The protein association of both tamalin and c-Src, as determined by truncation analysis of mGluR1 in COS-7 cells, occurred at the carboxyl-terminal tail of mGluR1. Mutation analysis of tyrosine with phenylalanine in COS-7 cells revealed that paired tyrosines at the ITAM sequence of tamalin are phosphorylated preferentially by c-Src and Fyn, and this phosphorylation can recruit Syk kinase and enables it to be phosphorylated by the Src family kinases. The phosphorylated tyrosines at the ITAM sequence of tamalin were highly susceptible to dephosphorylation by protein-tyrosine phosphatases in COS-7 cells. Importantly, tamalin was endogenously phosphorylated and associated with Syk in retinoic acid-treated P19 embryonal carcinoma cells that undergo neuron-like differentiation. The present investigation demonstrates that tamalin is a novel signaling molecule that possesses a PDZ domain and a PDZ binding motif and mediates Syk signaling in an ITAM-based fashion.

Negative cooperativity of glutamate binding in the dimeric metabotropic glutamate receptor subtype 1.

Metabotropic glutamate receptor (mGluR) subtype 1 is a Class III G-protein-coupled receptor that is mainly expressed on the post-synaptic membrane of neuronal cells. The receptor has a large N-terminal extracellular ligand binding domain that forms a homodimer, however, the intersubunit communication of ligand binding in the dimer remains unknown. Here, using the intrinsic tryptophan fluorescence change as a probe for ligand binding events, we examined whether allosteric properties exist in the dimeric ligand binding domain of the receptor. The indole ring of the tryptophan 110, which resides on the upper surface of the ligand binding pocket, sensed the ligand binding events. From saturation binding curves, we have determined the apparent dissociation constants (K(0.5)) of representative agonists and antagonists for this receptor (3.8, 0.46, 40, and 0.89 microm for glutamate, quisqualate, (S)-alpha-methyl-4-carboxyphenylglycine ((S)-MCPG), and (+)-2-methyl-4-carboxyphenylglycine (LY367385), respectively). Calcium ions functioned as a positive modulator for agonist but not for antagonist binding (K(0.5) values were 1.3, 0.21, 59, and 1.2 microm for glutamate, quisqualate, (S)-MCPG, and LY367385, respectively, in the presence of 2.0 mm calcium ion). Moreover, a Hill analysis of the saturation binding curves revealed the strong negative cooperativity of glutamate binding between each subunit in the dimeric ligand binding domain. As far as we know, this is the first direct evidence that the dimeric ligand binding domain of mGluR exhibits intersubunit cooperativity of ligand binding.

The nuclear bile acid receptor FXR is activated by PGC-1alpha in a ligand-dependent manner.

The nuclear bile acid receptor FXR (farnesoid X receptor) is one of the key factors that suppress bile acid biosynthesis in the liver. PGC-1alpha [PPARgamma (peroxisome-proliferator-activated receptor gamma) co-activator-1alpha] is known to control energy homoeostasis in adipose tissue, skeletal muscle and liver. We performed cell-based reporter assays using the expression system of a GAL4-FXR chimaera, the ligand-binding domain of FXR fused to the DNA-binding domain of yeast GAL4, to find the co-activators for FXR. We found that the transcriptional activation of a reporter plasmid by a GAL4-FXR chimaera was strongly enhanced by PGC-1alpha, in a ligand-dependent manner. Transcriptional activation of the SHP (small heterodimer partner) gene by the FXR-RXRalpha (retinoid X receptor alpha) heterodimer was also enhanced by PGC-1alpha in the presence of CDCA (chenodeoxycholic acid). Co-immunoprecipitation and pull-down studies using glutathione S-transferase-PGC-1alpha fusion proteins revealed that the ligand-binding domain of FXR binds PGC-1alpha in a ligand-influenced manner both in vivo and in vitro. Furthermore, our studies revealed that SHP represses its own transcription, and the addition of excess amounts of PGC-1alpha can overcome the inhibitory effect of SHP. These observations indicate that PGC-1alpha mediates the ligand-dependent activation of FXR and transcription of SHP gene.

Structure of the metabotropic glutamate receptor.

In the twelve years since the molecular elucidation of the metabotropic glutamate receptor subtype 1, a class III family of G-protein-coupled receptors has emerged; members of this family include the calcium-sensing receptor, the GABA(B) receptor, some odorant receptors and some taste receptors. Atomic structures of the ligand-binding core of the original metabotropic glutamate receptor 1 obtained using X-ray crystallography provide a foundation for determining the initial receptor activation of this important family of G-protein-coupled receptors.

Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor gamma coactivator-1 alpha. A possible link between xenobiotic response and nutritional state.

In contrast to the classical nuclear receptors, the constitutive androstane receptor (CAR) is transcriptionally active in the absence of ligand. In the course of searching for the mediator of CAR activation, we found that ligand-independent activation of CAR was achieved in cooperation with the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha). PGC-1 beta, a PGC-1 alpha homologue, also activated CAR to less of an extent than PGC-1 alpha. Coexpression of the ligand-binding domain of a heterodimerization partner, retinoid X receptor alpha, enhanced the PGC-1 alpha-mediated activation of CAR, although it had a weak effect on the basal activity of CAR in the absence of PGC-1 alpha. Both the N-terminal region, with the LXXLL motif, and the C-terminal region, with a serine/arginine-rich domain (RS domain), in PGC-1 alpha were required for full activation of CAR. Pull-down experiments using recombinant proteins revealed that CAR directly interacted with both the LXXLL motif and the RS domain. Furthermore, we demonstrated that the RS domain of PGC-1 alpha was required for CAR localization at nuclear speckles. These results indicate that PGC-1 alpha mediates the ligand-independent activation of CAR by means of subnuclear targeting through the RS domain of PGC-1 alpha.