ABSTRACT
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. Binding of mutant monomeric and native dimerizable IL-8 was investigated by NMR spectroscopy and isothermal titration calorimetry. Dendritic oligomerization of GAG increased the binding affinity of both monomeric and dimeric IL-8. Monomeric IL-8 bound to monomeric and dimeric GAG with KD values of 7.3 and 0.108â µM, respectively. The effect was less pronounced for dimerizable wild-type IL-8, for which GAG dimerization improved the affinity from 34 to 5â nM. Binding of dimeric IL-8 to oligomeric GAG was limited by steric crowding effects, strongly reducing the affinity of subsequent binding events. In conclusion, the strongest effect of GAG oligomerization was the amplified binding of IL-8 monomers, which might concentrate monomeric protein in the extracellular matrix and thus promote protein dimerization under physiological conditions.
Subject(s)
Glycosaminoglycans , Interleukin-8 , Glycosaminoglycans/chemistry , Dimerization , Interleukin-8/chemistry , Interleukin-8/metabolism , Proteoglycans , Protein BindingABSTRACT
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 (lo ) domains in artificial phase-separated membranes. Here, we synthesized globotriaosylceramide (Gb3 ) 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). Using phase-separated supported lipid bilayers on mica surfaces doped with four different photo-Gb3 molecules, we found by fluorescence microscopy and atomic force microscopy that liquid disordered (ld ) domains were formed within lo domains upon trans-cis photo-isomerization. The fraction and size of these ld domains were largest for Gb3 molecules with the azobenzene group at the end of the fatty acid. We further investigated the impact of domain reorganization on the interaction of the B-subunits of STx with the photo-Gb3 . Fluorescence and atomic force micrographs clearly demonstrated that STxB binds to the lo phase if Gb3 is in the trans-configuration, whereas two STxB populations are formed if the photo-Gb3 is switched to the cis-configuration highlighting the idea of manipulating lipid-protein interactions with a light stimulus.
Subject(s)
Lipid Bilayers , Shiga Toxin , Shiga Toxin/metabolism , Isomerism , Lipid Bilayers/metabolism , Fatty AcidsABSTRACT
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 15N-NMR and quantitative determination of 1H-13C 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 0.57 for the Cα sites and lower in the side chains (0.37 for the CH2 and 0.18 for the CH3 groups). Different NMR excitation schemes identify relatively rigid and also dynamic segments of the molecule. In monounsaturated membranes composed of longer lipid chains, Y1R is more rigid, attributed to a higher hydrophobic thickness of the lipid membrane. The presence of an antagonist or NPY has little influence on the amplitude of motions, whereas the addition of agonist and arrestin led to a pronounced rigidization. To investigate Y1R dynamics with site resolution, we conducted extensive all-atom MD simulations of the apo and antagonist-bound state. In each state, three replicas with a length of 20 µs (with one exception, where the trajectory length was 10 µs) were conducted. In these simulations, order parameters of each residue were determined and showed high values in the transmembrane helices, whereas the loops and termini exhibit much lower order. The extracellular helix segments undergo larger amplitude motions than their intracellular counterparts, whereas the opposite is observed for the loops, Helix 8, and termini. Only minor differences in order were observed between the apo and antagonist-bound state, whereas the time scale of the motions is shorter for the apo state. Although these relatively fast motions occurring with correlation times of ns up to a few µs have no direct relevance for receptor activation, it is believed that they represent the prerequisite for larger conformational transitions in proteins.
Subject(s)
Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Protein Conformation , Receptors, Neuropeptide Y/chemistry , Drug Discovery , Magnetic Resonance Spectroscopy/methods , Nuclear Magnetic Resonance, Biomolecular , Receptors, Neuropeptide Y/antagonists & inhibitors , Recombinant Proteins/chemistry , Structure-Activity RelationshipABSTRACT
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. The long chain lignoceryl-Gb3 showed a rather unusual order parameter profile of the acyl chain, which flattens out for the last â¼6 methylene segments. Such an odd chain conformation can be explained by partial chain interdigitation and/or a very fluid midplane region of the membrane. Possibly, the Gb3 molecules may thus preferentially be localized at the lo/ld phase boundary. In contrast, the short chain palmitoyl-Gb3 was well associated with the PSM/cholesterol-rich lo phase. Gb3 molecules act as membrane receptors for the Shiga toxin (STx) produced by Shigella dysenteriae and by enterohemorrhagic strains of Escherichia coli (EHEC). The B-subunits of STx (STxB) forming a pentameric structure were produced recombinantly and incubated with the membrane mixtures leading to alterations in the lipid packing properties and lateral organization of the membranes. Typically, STxB binding led to a decrease in lipid chain order in agreement with partial immersion of protein segments into the lipid-water interface of the membrane. In the presence of STxB, Gb3 preferentially partitioned into the lo membrane phase. In particular the short acyl chain palmitoyl-Gb3 showed very similar chain order parameters to PSM. In the presence of STxB, all lipid species showed isotropic contributions to the 2H NMR powder spectra; this was most pronounced for the Gb3 molecules. Such isotropic contributions are caused by highly curved membrane structures, which have previously been detected as membrane invaginations in fluorescence microscopy. Our analysis estimated that STxB induced highly curved membrane structures with a curvature radius of less than â¼10 nm likely related to the insertion of STxB segments into the lipid-water interface of the membrane.
Subject(s)
Lipid Bilayers/chemistry , Lipids/chemistry , Magnetic Resonance Spectroscopy , Shiga Toxin/chemistry , Protein BindingABSTRACT
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. This information combined with assignment of the secondary structure of ghrelin in its receptor-bound state was incorporated into Rosetta using an approach that accounts for flexible binding partners. The NMR data and models revealed an extended binding surface that was confirmed via mutagenesis. Our results agree with a growing evidence of peptides interacting via two sites at G protein-coupled receptors.
Subject(s)
Ghrelin/chemistry , Ghrelin/metabolism , Receptors, Ghrelin/metabolism , Animals , Binding Sites , COS Cells , Chlorocebus aethiops , HEK293 Cells , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Mutagenesis, Site-Directed , Protein Binding , Protein ConformationABSTRACT
Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . 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 Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R) 4 . A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.
Subject(s)
Arginine/analogs & derivatives , Dihydropyridines/chemistry , Dihydropyridines/metabolism , Diphenylacetic Acids/chemistry , Diphenylacetic Acids/metabolism , Neuropeptide Y/metabolism , Phenylurea Compounds/chemistry , Phenylurea Compounds/metabolism , Receptors, Neuropeptide Y/antagonists & inhibitors , Receptors, Neuropeptide Y/chemistry , Arginine/chemistry , Arginine/metabolism , Arginine/pharmacology , Binding Sites , Crystallography, X-Ray , Dihydropyridines/pharmacology , Diphenylacetic Acids/pharmacology , Humans , Inositol Phosphates/metabolism , Ligands , Molecular Docking Simulation , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , Neuropeptide Y/chemistry , Neuropeptide Y/pharmacology , Nuclear Magnetic Resonance, Biomolecular , Phenylurea Compounds/pharmacology , Protein Binding , Receptors, Neuropeptide Y/agonists , Receptors, Neuropeptide Y/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
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. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40-50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.
Subject(s)
Lipid Bilayers/chemistry , Magnetic Resonance Spectroscopy , Receptors, Ghrelin/metabolism , Dimyristoylphosphatidylcholine/chemistry , Ghrelin/metabolism , Humans , Phosphatidylcholines/chemistry , Receptors, Ghrelin/agonists , Receptors, Ghrelin/chemistry , Recombinant Proteins/metabolismABSTRACT
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 neuropeptideâ Y (NPY) bound to its human G-protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specific isotope-labeled NPY in complex with inâ vitro folded Y2R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T(32) to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.
Subject(s)
Neuropeptide Y/metabolism , Receptors, Neuropeptide Y/metabolism , Amino Acid Sequence , Binding Sites , Humans , Molecular Docking Simulation , Molecular Sequence Data , Neuropeptide Y/chemistry , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Secondary , Receptors, Neuropeptide Y/chemistryABSTRACT
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. But this strategy can only successfully be applied if the subsequent in vitro folding results in a high yield of active receptors and if this fraction can be isolated from the nonactive receptors in a homogeneous form. Here, we followed that strategy to provide large quantities of the human neuropeptide Y receptor type 2 and determined the folding yield before and after ligand affinity chromatography using a radioligand binding assay. Directly after folding, we achieved a proportion of ~25% active receptor. This value could be increased to ~96% using ligand affinity chromatography. Thus, a very homogeneous sample of the Y2 receptor could be prepared that exhibited a K(D) value of 0.1 ± 0.05 nM for the binding of polypeptide Y, which represents one of the natural ligands of the Y2 receptor.