Dimerization and Crowding in the Binding of Interleukin 8 to Dendritic Glycosaminoglycans as Artificial Proteoglycans.

The interactions of glycosaminoglycans (GAG) with proteins of the extracellular matrix govern and regulate complex physiological functions including cellular growth, immune response, and inflammation. Repetitive presentation of GAG binding motifs, as found in native proteoglycans, might enhance GAG-protein binding through multivalent interactions. Here, we report the chemical synthesis of dendritic GAG oligomers constructed of nonasulfated hyaluronan tetrasaccharides for investigating the binding of the protein chemokine interleukin 8 (IL-8) to artificial, well-defined proteoglycan architectures.

Optical Manipulation of Gb Enriched Lipid Domains: Impact of Isomerization on Gb -Shiga Toxin B Interaction.

The plasma membrane is a complex assembly of proteins and lipids that can self-assemble in submicroscopic domains commonly termed "lipid rafts", which are implicated in membrane signaling and trafficking. Recently, photo-sensitive lipids were introduced to study membrane domain organization, and photo-isomerization was shown to trigger the mixing and de-mixing of liquid-ordered (l ) domains in artificial phase-separated membranes. Here, we synthesized globotriaosylceramide (Gb ) glycosphingolipids that harbor an azobenzene moiety at different positions of the fatty acid to investigate light-induced membrane domain reorganization, and that serve as specific receptors for the protein Shiga toxin (STx).

The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation.

We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using N-NMR and quantitative determination of H-C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.

Shiga toxin binding alters lipid packing and the domain structure of Gb-containing membranes: a solid-state NMR study.

We studied the influence of globotriaosylceramide (Gb3) lipid molecules on the properties of phospholipid membranes composed of a liquid ordered (lo)/liquid disordered (ld) phase separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/N-palmitoyl-d-erythro-sphingosylphosphorylcholine (PSM)/cholesterol mixture (40/35/20, mol/mol/mol) supplemented with 5 mol% of either short acyl chain palmitoyl-Gb3 or long acyl chain lignoceryl-Gb3 using 2H solid-state NMR spectroscopy. To this end, both globotriaosylceramides were chemically synthesized featuring a perdeuterated lipid acyl chain. The solid-state 2H NMR spectra support the phase separation into a POPC-rich ld phase and a PSM/cholesterol-rich lo phase.

Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor.

The peptide ghrelin targets the growth hormone secretagogue receptor 1a (GHSR) to signal changes in cell metabolism and is a sought-after therapeutic target, although no structure is known to date. To investigate the structural basis of ghrelin binding to GHSR, we used solid-state nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, and Rosetta modeling. The use of saturation transfer difference NMR identified key residues in the peptide for receptor binding beyond the known motif.

Structural basis of ligand binding modes at the neuropeptide Y Y receptor.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y, Y, Y and Y receptors, with different affinity and selectivity . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y receptor (YR) .

Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes.

The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli.

Unwinding of the C-Terminal Residues of Neuropeptide Y is critical for Y₂ Receptor Binding and Activation.

Despite recent breakthroughs in the structural characterization of G-protein-coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double-cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G-protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specific isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide.

Assessment of a fully active class A G protein-coupled receptor isolated from in vitro folding.

We provide a protocol for the preparation of fully active Y2 G protein-coupled receptors (GPCRs). Although a valuable target for pharmaceutical research, information about the structure and dynamics of these molecules remains limited due to the difficulty in obtaining sufficient amounts of homogeneous and fully active receptors for in vitro studies. Recombinant expression of GPCRs as inclusion bodies provides the highest protein yields at lowest costs.