Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.
Ontology highlight
ABSTRACT: BACKGROUND AND PURPOSE: The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. EXPERIMENTAL APPROACH: The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. KEY RESULTS: Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. CONCLUSIONS: These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.
Project description:Thymidylate synthase (TS) catalyzes the production of the nucleotide dTMP from deoxyuridine monophosphate (dUMP), making the enzyme necessary for DNA replication and consequently a target for cancer therapeutics. TSs are homodimers with active sites separated by ∼30 Å. Reports of half-the-sites activity in TSs from multiple species demonstrate the presence of allosteric communication between the active sites of this enzyme. A simple explanation for the negative allosteric regulation occurring in half-the-sites activity would be that the two substrates bind with negative cooperativity. However, previous work on Escherichia coli TS revealed that dUMP substrate binds without cooperativity. To gain further insight into TS allosteric function, binding cooperativity in human TS is examined here. Isothermal titration calorimetry and two-dimensional lineshape analysis of NMR titration spectra are used to characterize the thermodynamics of dUMP binding, with a focus on quantification of cooperativity between the two substrate binding events. We find that human TS binds dUMP with ∼9-fold entropically driven positive cooperativity (ρITC = 9 ± 1, ρNMR = 7 ± 1), in contrast to the apparent strong negative cooperativity reported previously. Our work further demonstrates the necessity of globally fitting isotherms collected under various conditions, as well as accurate determination of binding competent protein concentration, for calorimetric characterization of homotropic cooperative binding. Notably, an initial curvature of the isotherm is found to be indicative of positively cooperative binding. Two-dimensional lineshape analysis NMR is also found to be an informative tool for quantifying binding cooperativity, particularly in cases in which bound intermediates yield unique resonances.
Project description:Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the "high-affinity" and "low-affinity" classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.
Project description:The epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, migration, and differentiation, and aberrant EGFR signaling is implicated in a variety of cancers. EGFR signaling is triggered by extracellular ligand binding, which promotes EGFR dimerization and activation. Ligand-binding measurements are consistent with a negatively cooperative model in which the ligand-binding affinity at either binding site in an EGFR dimer is weaker when the other site is occupied by a ligand. This cooperativity is widely believed to be central to the effects of ligand concentration on EGFR-mediated intracellular signaling. Although the extracellular portion of the human EGFR dimer has been resolved crystallographically, the crystal structures do not reveal the structural origin of this negative cooperativity, which has remained unclear. Here we report the results of molecular dynamics simulations suggesting that asymmetrical interactions of the two binding sites with the membrane may be responsible (perhaps along with other factors) for this negative cooperativity. In particular, in our simulations the extracellular domains of an EGFR dimer spontaneously lay down on the membrane in an orientation in which favorable membrane contacts were made with one of the bound ligands, but could not be made with the other. Similar interactions were observed when EGFR was glycosylated, as it is in vivo.
Project description:The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, meaning that the human P2X7R (hP2X7R) is an attractive therapeutic target. The crystal structures of the zebrafish P2X4R in the closed and ATP-bound open states provide an unprecedented opportunity for structure-guided identification of new ligands. The present study performed virtual screening of ∼100,000 structurally diverse compounds against the ATP-binding pocket in the hP2X7R. This identified three compounds (C23, C40 and C60) out of 73 top-ranked compounds by testing against hP2X7R-mediated Ca(2+) responses. These compounds were further characterised using Ca(2+) imaging, patch-clamp current recording, YO-PRO-1 uptake and propidium iodide cell death assays. All three compounds inhibited BzATP-induced Ca(2+) responses concentration-dependently with IC50s of 5.1±0.3μM, 4.8±0.8μM and 3.2±0.2μM, respectively. C23 and C40 inhibited BzATP-induced currents in a reversible and concentration-dependent manner, with IC50s of 0.35±0.3μM and 1.2±0.1μM, respectively, but surprisingly C60 did not affect BzATP-induced currents up to 100μM. They suppressed BzATP-induced YO-PRO-1 uptake with IC50s of 1.8±0.9μM, 1.0±0.1μM and 0.8±0.2μM, respectively. Furthermore, these three compounds strongly protected against ATP-induced cell death. Among them, C40 and C60 exhibited strong specificity towards the hP2X7R over the hP2X4R and rP2X3R. In conclusion, our study reports the identification of three novel hP2X7R antagonists with micromolar potency for the first time using a structure-based approach, including the first P2X7R antagonist with preferential inhibition of large pore formation.
Project description:Background and purposeConventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy.Experimental approachFiltration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor.Key resultsAgonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins.Conclusions and implicationsThese findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding.
Project description:Preorganization is a basic design principle used by nature that allows for synergistic pathways to be expressed. Herein, a full account of the conceptual and experimental development from randomly distributed functionalities to a convergent arrangement that facilitates cooperative binding is given, thus conferring exceptional affinity toward the analyte of interest. The resulting material with chelating groups populated adjacently in a spatially locked manner displays up to two orders of magnitude improvement compared to a random and isolated manner using uranium sequestration as a model application. This adsorbent shows exceptional extraction efficiencies, capable of reducing the uranium concentration from 5 ppm to less than 1 ppb within 10 min, even though the system is permeated with high concentrations of competing ions. The efficiency is further supported by its ability to extract uranium from seawater with an uptake capability of 5.01 mg g-1, placing it among the highest-capacity seawater uranium extraction materials described to date. The concept presented here uncovers a new paradigm in the design of efficient sorbent materials by manipulating the spatial distribution to amplify the cooperation of functions.
Project description:The binding of EGF induces dimerization of its receptor, leading to the stimulation of its intracellular tyrosine kinase activity. Kinase activation occurs within the context of an asymmetric dimer in which one kinase domain serves as the activator for the other kinase domain but is not itself activated. How ligand binding is related to the formation and dynamics of this asymmetric dimer is not known. The binding of EGF to its receptor is negatively cooperative--that is, EGF binds with lower affinity to the second site on the dimer than to the first site on the dimer. In this study, we analyzed the binding of (125)I-EGF to a series of EGF receptor mutants in the intracellular juxtamembrane domain and demonstrate that the most membrane-proximal portion of this region plays a significant role in the genesis of negative cooperativity in the EGF receptor. The data are consistent with a model in which the binding of EGF to the first site on the dimer induces the formation of one asymmetric kinase dimer. The binding of EGF to the second site is required to disrupt the initial asymmetric dimer and allow the formation of the reciprocal asymmetric dimer. Thus, some of the energy of binding to the second site is used to reorient the first asymmetric dimer, leading to a lower binding affinity and the observed negative cooperativity.
Project description:P2X7 receptors are important in the regulation of inflammatory responses and immune responses to intracellular pathogens such as Mycobacterium tuberculosis and Toxoplasma gondii. Enhancement of P2X7 receptor responses may be useful in pathogen clearance particularly in individuals with defective microbial killing mechanisms. Ginsenosides from Panax ginseng have been discovered to act as positive allosteric modulators of P2X7. Here we describe a novel modulator binding site identified by computational docking located in the central vestibule of P2X7 involving S60, D318, and L320 in the lower body β-sheets lining the lateral portals. Potentiation of ATP-mediated responses by ginsenosides CK and Rd caused enhanced ionic currents, Ca2+ influx and YOPRO-1 uptake in stably transfected HEK-293 cells (HEK-hP2X7) plus enhanced cell death responses. Potentiation of ATP responses by CK and Rd was markedly reduced by mutations S59A, S60A, D318L and L320A supporting the proposed allosteric modulator binding site. Furthermore, mutation of the conserved residues S60 and D318 led to alterations in P2X7 response and a higher sensitivity to ATP in the absence of modulators suggesting residues in the connecting rods play an important role in regulating P2X7 gating. Identification of this novel binding site location in the central vestibule may also be relevant for structurally similar channels.
Project description:P2X receptors are widely distributed in the nervous system, and P2X7 receptors have roles in neuropathic pain and in the release of cytokines from microglia. They are trimeric membrane proteins, which open an integral ion channel when ligated by extracellular ATP. This channel is preferentially permeable to small cations (sodium, potassium, calcium) but also allows permeation of larger cations such as N-methyl-d-glucamine. ATP also leads to entry of fluorescent dyes in many cells expressing P2X7 receptors, but controversy persists as to whether such large molecules pass directly through the open ion channel or enter the cell by a different route. We measured cellular fluorescence and membrane currents in individual human embryonic kidney cells expressing rat P2X7 receptors. Introduction of positive side chains by mutagenesis into the inner half of the pore-forming second transmembrane domain of the receptor (T348K, D352N, D352K) increased relative permeability of chloride ions. It also promoted entry of the large (>1 nm) negative dye fluorescein-5-isothiocyanate while decreasing entry of the structurally similar but positive dye ethidium. Furthermore, larger cysteine-reactive methanethiosulfonates [sulforhodamine-methanethiosulfonate and 2-((biotinoyl)amino)ethyl methanethiosulfonate] reduced both ATP-evoked currents and dye entry when applied to open P2X7[G345C] receptors. The results demonstrate that the open channel of the P2X7 receptor can allow passage of molecules with sizes up to 1.4 nm.
Project description:P2X(7) receptors (P2X(7)R) are ATP-gated calcium-permeable cationic channels structurally unique among the P2X family by their much longer intracellular C-terminal tail. P2X(7)Rs show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X(7)R resulted from a Ca(2+)-calmodulin-dependent process and a distinct calcium-independent process. However, P2X(7)Rs show striking species differences; thus, this study compared the properties of ATP-evoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X(7)R as well as rat/human, human/rat chimeric, and mutated P2X(7)Rs. Facilitation at the human P2X(7)R was 5-fold slower than at the rat P2X(7)R. Facilitation did not resulting from an increase of receptor addressing the plasma membrane. We found the human P2X(7)R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C terminus of rat P2X(7)R. Replacement of three critical residues of this binding domain from the rat into the human P2X(7)R (T541I, C552S, and G559V) reconstituted the Ca(2+)-calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X(7)Rs. The absence of Ca(2+)-dependent facilitation at the human P2X(7)R may represent a protective adaptation of the innate immune response in which P2X(7)R plays significant roles.