RNA substrate binding site in the catalytic core of the Tetrahymena ribozyme.

In catalysis by group I introns, the helix (P1) containing the RNA cleavage site must be positioned next to the guanosine binding site. We have identified a conserved adenine in the catalytic core that contributes to the stability of this arrangement and propose that it accepts a hydrogen bond from a specific 2'-OH in P1. Such base-backbone tertiary interactions may be generally important to the organization of RNA tertiary structure.     

The guanosine binding site of the Tetrahymena ribozyme

The self-splicing Group I introns have a highly specific binding site for the substrate guanosine. Mutant versions of the Tetrahymena ribozyme have been used in combination with guanosine analogues to identify the nucleotide in the ribozyme that is primarily responsible for recognition of the guanine base.     

Differential localization of type I and type II benzodiazepine binding sites in substantia nigra

A number of studies have suggested the existence of multiple benzodiazepine binding sites in the brain. We have recently reported the physical separation of two apparent benzodiazepine binding site subtypes, the pharmacological properties, and distribution in tissue sections of which correspond to the putative type I and type II sites. Benzodiazepine and gamma-aminobutyric acid (GABA) receptors have been shown to interact, and lesions of the GABAergic striatonigral pathway, which lead to GABA supersensitivity, both increase the numbers of GABA binding sites and enhance GABA-stimulated benzodiazepine binding. We demonstrate here that degeneration of striatonigral fibres increases the density of putative type I benzodiazepine binding sites in the substantia nigra and decreases the density of the putative type II sites. This suggests that type I sites that increase after denervation are postsynaptic, whereas the type II sites reduced by the lesion may be localized to axons or terminals of the striatonigral pathways.     

The B-cell binding site on human immunoglobulin E

Immunoglobulin E comprises the main immunoglobulin class associated with allergy. Its multifarious activities are mediated by two types of Fc receptors found on different cell populations, Fc epsilon R1 on mast cells and basophils, and Fc epsilon R2 on inflammatory cells (monocytes, eosinophils and platelets) and B lymphocytes. Recombinant epsilon-chain fragments synthesized in Escherichia coli have provided the means of mapping the receptor-binding sites on human IgE, and blocking IgE-receptor interactions. We have previously shown that the Fc epsilon R1 binding site is contained within a sequence (Gln 301-Arg 376) spanning the C epsilon 2 and C epsilon 3 domains. Here we show that Fc epsilon R2 can recognize a motif in the C epsilon 3 domain that is formed on dimerization of one or both of the flanking (C epsilon 2 and C epsilon 4) domains. Glycosylation of IgE is not required for the activity of either receptor.     

Antidepressant binding site in a bacterial homologue of neurotransmitter transporters

Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 A above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.     

The mast cell binding site on human immunoglobulin E

Antibodies of the immunoglobulin E isotype sensitize mast cells and basophils for antigen-induced mediator release by binding through the Fc portion to a high-affinity receptor (Fc epsilon R1, Ka = 10(9)M-1) on the cell surface causing the clinical manifestations of type I hypersensitivity. As the amino acid sequence of the human epsilon chain is now known, attempts have been made to map the Fc epsilon R1 binding site on IgE to a fragment smaller than Fc epsilon using proteolytic cleavage products, none of which proved to be active. Cleavage between the C epsilon 2 and C epsilon 3 domains released two inactive fragments, suggesting that the junction between these segments could be important in receptor binding. This region is protected against protease digestion in the rat IgE complex with the receptor of rat basophilic leukaemia cells. Here we report the mapping of the mast cell receptor binding site on human IgE to a sequence of 76 amino acids at the C epsilon 2/C epsilon 3 junction. Recombinant peptides containing this sequence inhibit passive sensitization of skin mast cells in vivo and sensitize mast cells to degranulation by anti-IgE in vitro almost as efficiently as a myeloma IgE. Fragments containing the separate domains are inactive. Additional sequences are required for rapid assembly of fragments into disulphide-linked dimers, suggesting that a single chain can form the active site. In a three-dimensional model of the human Fc epsilon, the two identical segments are far apart. Each folds to generate a cleft between the C epsilon 2 and C epsilon 3 domains on the surface of the Fc epsilon. The docking of IgE on to mast cells could take place within this cleft.     

Identification of a CD4 binding site on the beta 2 domain of HLA-DR molecules.

The CD4 and CD8 molecules are transmembrane glycoproteins expressed by functionally distinct subsets of mature T cells. CD4+ and CD8+ T cells recognize antigens on major histocompatibility complex (MHC) class II-bearing and class I-bearing target cells respectively. The ability of monoclonal antibodies against CD4 and CD8 to block antigen recognition by T cells, as well as cell-cell adhesion assays, indicate that CD4 and CD8 bind to nonpolymorphic determinants of class II or class I MHC. Here we demonstrate that soluble recombinant HLA-DR4 molecules from insect cells and HLA-DR-derived peptides bind to immobilized recombinant soluble CD4. CD4 binds recombinant soluble DR4 heterodimers, as well as the soluble DR4-beta chain alone. Furthermore, two out of twelve DR4-beta peptides could interact specifically with CD4. These findings show that CD4 interacts with a region of MHC class II molecules analogous to a previously identified loop in class I MHC proteins that binds CD8 (refs 8, 9).     

The binding site for C1q on IgG

In humoral defence, pathogens are cleared by antibodies acting as adaptor molecules: they bind to antigen and trigger clearance mechanisms such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and complement lysis. The first step in the complement cascade is the binding of C1q to the antibody. There are six heads on C1q, connected by collagen-like stems to a central stalk, and the isolated heads bind to the Fc portion of antibody rather weakly, with an affinity of 100 microM (ref. 3). Binding of antibody to multiple epitopes on an antigenic surface, aggregates the antibody and this facilitates the binding of several C1q heads, leading to an enhanced affinity of about 10 nM (ref. 1). Within the Fc portion of the antibody, C1q binds to the CH2 domain. The interaction is sensitive to ionic strength, and appears to be highly conserved throughout evolution as C1q reacts with IgG from different species (for example see ref. 8). By systematically altering surface residues in the mouse IgG2b isotype, we have localized the binding site for C1q to three side chains, Glu 318, Lys 320 and Lys 322. These residues are relatively conserved in other antibody isotypes, and a peptide mimic of this sequence is able to inhibit complement lysis. We propose that this sequence motif forms a common core in the interactions of IgG and C1q.     

A functional correlate for the dihydropyridine binding site in rat brain

Calcium channels, controlling the influx of extracellular Ca2+ and hence neurotransmitter release, exist in the brain. However, drugs classed as calcium antagonists and which inhibit Ca2+ entry through voltage-activated Ca2+ channels in heart and smooth muscle, seem not to affect any aspect of neuronal function in the brain at pharmacologically relevant concentrations. Yet the dihydropyridine calcium antagonists (for example, nitrendipine) bind stereospecifically with high affinity to a recognition site on brain-cell membranes thought to represent the Ca2+ channel and consequently, the physiological relevance of these sites has been questioned. However, activation of voltage-dependent Ca2+ channels can increase cytoplasmic Ca2+ and neurotransmitter release in neuronal tissue. We show here that Bay K8644, a dihydropyridine Ca2+-channel activator, can augment K+-stimulated release of serotonin from rat frontal cortex slices and that these effects can be antagonized by low concentrations of calcium antagonists. As 3H-dihydropyridine binding to cortical membrane preparations resembles the binding in heart and smooth muscle where there are good functional correlates we conclude that the dihydropyridine binding sites in the brain represent functional Ca2+ channels that can be unmasked under certain circumstances.     

Correlation between benzodiazepine receptor occupation and anticonvulsant effects of diazepam.

The benzodiazepines are potent anticonvulsants for a wide variety of experimental and clinical seizure disorders. The demonstration of saturable, high-affinity and stereospecific binding sites for the benzodiazepines in the mammalian central nervous system suggests the presence of pharmacological receptors mediating the anticonvulsant properties of these compounds. The good correlation between the anticonvulsant potencies of a series of benzodiazepines and their ability to inhibit 3H-diazepam binding in vitro further supports this hypothesis, but evidence for a direct interaction between benzodiazepines and their receptors, and a subsequent inhibition of seizure activity (or elevation of seizure threshold) is lacking. Recent reports from our laboratory and others have demonstrated the feasibility of labelling benzodiazepine receptors in vivo following parental administration of tritiated benzodiazepine. This technique permits one to study the relationship between the anticonvulsant activity of the benzodiazepines in vivo and the number of 'drug-occupied' receptors in vitro. We now report that there is an excellent correlation between benzodiazepine receptor occupancy by diazepam and protection against pentylenetetrazol-induced seizures. Furthermore, these results demonstrate that only a small fraction of benzodiazepine receptors need be occupied to produce a complete anticonvulsant effect.