Studies using low-resolution fiber diffraction, electron microscopy, and atomic force microscopy on various amyloid fibrils indicate that the misfolded conformers must be modular, compact, and adopt a cross- structure. In an earlier study, we used electron crystallography to delineate molecular models of the N-terminally truncated, disease-causing isoform (PrP Sc ) of the prion protein, designated PrP 27-30, which polymerizes into amyloid fibrils, but we were unable to choose between a trimeric or hexameric arrangement of right-or left-handed -helical models. From a study of 119 all- folds observed in globular proteins, we have now determined that, if PrP Sc follows a known protein fold, it adopts either a -sandwich or parallel -helical architecture. With increasing evidence arguing for a parallel -sheet organization in amyloids, we contend that the sequence of PrP is compatible with a parallel left-handed -helical fold. Left-handed -helices readily form trimers, providing a natural template for a trimeric model of PrP Sc . This trimeric model accommodates the PrP sequence from residues 89 -175 in a -helical conformation with the C terminus (residues 176 -227), retaining the disulfide-linked ␣-helical conformation observed in the normal cellular isoform. In addition, the proposed model matches the structural constraints of the PrP 27-30 crystals, positioning residues 141-176 and the N-linked sugars appropriately. Our parallel left-handed -helical model provides a coherent framework that is consistent with many structural, biochemical, immunological, and propagation features of prions. Moreover, the parallel left-handed -helical model for PrP Sc may provide important clues to the structure of filaments found in some other neurodegenerative diseases.
Clones encoding a new human P2Y receptor, provisionally called P2Y 11 , have been isolated from human placenta complementary DNA and genomic DNA libraries. The 1113-base pair open reading frame is interrupted by one intron. The P2Y 11 receptor is characterized by considerably larger second and third extracellular loops than the subtypes described so far. The deduced amino acid sequence exhibits 33% amino acid identity with the P2Y 1 receptor, its closest homolog. Northern blot analysis detected human P2Y 11 receptor messenger RNA in spleen and HL-60 cells. The level of P2Y 11 transcripts was strongly increased in these cells after granulocyte differentiation induced by retinoic acid or dimethyl sulfoxide. The new receptor was stably expressed in 1321N1 astrocytoma and CHO-K1 cells, where it couples to the stimulation of both the phosphoinositide and adenylyl cyclase pathways, a unique feature among the P2Y family. The rank order of agonists potency was: ATP > 2-methylthio-ATP >>> ADP, whereas UTP and UDP were inactive, indicating that it behaves as a selective purinoceptor.An impressive number of P 2 receptor subtypes has been cloned since 1993. A molecular nomenclature has been established in which G protein-coupled P 2 receptors are named P2Y, whereas P 2 receptors having an intrinsic ion channel activity are named P2X (1, 2). The P2Y family encompasses selective purinoceptors (the P2Y 1 receptor (3, 4) activated preferentially by ADP and ATP), nucleotide receptors responsive to both adenine and uracil nucleotides (the P2Y 2 receptor (5, 6) activated equipotently by ATP and UTP, and the P2Y 8 receptor (7) activated equally by all triphosphate nucleotides), and pyrimidinoceptors (the P2Y 3 (8) and P2Y 6 (9 -11) receptors activated preferentially by UDP, and the P2Y 4 receptor (11-13) activated preferentially by UTP). All these P2Y subtypes are coupled exclusively to the phosphoinositide pathway. The inclusion of other receptors (P2Y 5 (14) and P2Y 7 (15)) within the P2Y family remains controversial (16 -18). Here we report the cloning of a new human gene encoding a G protein-coupled receptor that behaves as a selective purinoceptor and is coupled to the stimulation of both adenylyl cyclase and phospholipase C. 6 A, all-trans retinoic acid, and 12-Otetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma. 2-Methylthio-ATP (2MeSATP), 2-methylthio-ADP (2MeSADP) and 8-(p-sulfophenyl) theophylline were from Research Biochemicals International (Natick, MA). Forskolin was purchased from Calbiochem. (Bierges, Belgium). Indomethacin and Me 2 SO were from Merck. Rolipram was a gift from the Laboratoires Jacques Logeais (Trappes, France). The HL-60 human cell line was obtained from the American Type Culture Collection (Rockville, MD). The human genomic DNA library was from Stratagene (La Jolla, CA). The human placenta cDNA library was kindly given by Prof. P. Chambon (Strasbourg, France). pEFIN3 is an expression vector developed by Euroscreen (Brussels, Belgium). Multiple human tissue Northern blot (MTN) were from ...
Lipids are emerging as key regulators of membrane protein structure and activity. Such effects can either be attributed to modification in bilayer properties (thickness, curvature and surface tension) or to binding of specific lipids to the protein surface. For G Protein-Coupled Receptors (GPCRs), the effect of phospholipids on receptor structure and activity remains poorly understood. Here we reconstituted purified β2-adrenergic receptor in High-Density-Lipoparticles to systematically characterize the effect of biologically relevant phospholipids on receptor activity. We observe that the lipid head-group type affects ligand binding (agonist and antagonist) and receptor activation. Specifically, phosphatidylgycerol markedly favors agonist binding and facilitates receptor activation while phosphatidylethanolamine favors antagonist binding and stabilizes the inactive state of the receptor. We then show that these effects can be recapitulated with detergent-solubilized lipids, demonstrating that the functional modulation occurs in the absence of a bilayer. Our data suggest that phospholipids act as direct allosteric modulators of GPCR activity.
In the past few years there has been a growth in the use of nano-particles for stabilizing lipid membranes with embedded proteins. These bionanoparticles provide a solution to the challenging problem of membrane protein isolation by maintaining a lipid bilayer essential to protein integrity and activity. We have described the use of an amphipathic polymer (Poly(styrene-co-maleic acid); SMA) to produce discoidal nanoparticles that contain a lipid bilayer with embedded protein. However the structure of the nanoparticle itself has not yet been determined. This leaves a major gap in understanding how the SMA stabilizes the encapsulated bilayer and how the bilayer relates physically and structurally to an unecapsulated lipid bilayer. In this paper we address this issue by describing the structure of the SMA Lipid Particle (SMALP) using data from small angle neutron scattering (SANS), electron microscopy (EM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC) and nuclear magnetic resonance spectroscopy (NMR). We show that the particle is disc shaped containing a polymer “bracelet” encircling the lipid bilayer. The structure and orientation of the individual components within the bilayer and polymer are determined showing that styrene moieties within SMA intercalate between the lipid acyl chains. The dimensions of the encapsulated bilayer are also determined and match those measured for a natural membrane. Taken together, the description of structure of the SMALP forms the foundation of future development and applications of SMALPs in membrane protein production and analysis.
P-glycoprotein (P-gp) is one of the best-known mediators of drug efflux-based multidrug resistance in many cancers. This validated therapeutic target is a prototypic, plasma membrane resident ATPBinding Cassette transporter that pumps xenobiotic compounds out of cells. The large, polyspecific drug-binding pocket of P-gp recognizes a variety of structurally unrelated compounds. The transport of these drugs across the membrane is coincident with changes in the size and shape of this pocket during the course of the transport cycle. Here, we present the crystal structures of three inward-facing conformations of mouse P-gp derived from two different crystal forms. One structure has a nanobody bound to the C-terminal side of the first nucleotide-binding domain. This nanobody strongly inhibits the ATP hydrolysis activity of mouse Pgp by hindering the formation of a dimeric complex between the ATP-binding domains, which is essential for nucleotide hydrolysis. Together, these inward-facing conformational snapshots of P-gp demonstrate a range of flexibility exhibited by this transporter, which is likely an essential feature for the binding and transport of large, diverse substrates. The nanobody-bound structure also reveals a unique epitope on P-gp.
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