Identification by cross-linking of a neuronal acceptor protein for dendrotoxin, a convulsant polypeptide.

Dendrotoxin, a lijow molecular weight protein from the venom of Dendroaspis angusticeps, is known to be a potent convulsant that attenuates one type of voltage-sensitive K+ channel in guinea-pig hippocampus. A biologically active preparation of 125I-labelled dendrotoxin has been cross-linked to its high-affinity protein acceptor in synaptic plasma membranes from rat cerebral cortex. On SDS gel electrophoresis, a complex with a Mr of 72,000 was observed which, assuming one toxin molecule is attached, yields an apparent size of 65,000 for this subunit of the acceptor. Unlike dendrotoxin, low concentrations of beta-bungarotoxin, another pre-synaptically active toxin, do not inhibit its labelling.

Interactions between discrete neuronal membrane binding sites for the putative K+-channel ligands beta-bungarotoxin, dendrotoxin and mast-cell-degranulating peptide.

1. beta-Bungarotoxin, a presynaptically active neurotoxin from the venom of Bungarus multicinctus, was radiolabelled with 125I and its binding to synaptic membranes from rat brain was analyzed. The interaction of these binding sites with those for dendrotoxin (a convulsant polypeptide from mamba venom) and mast-cell-degranulating peptide (from bee venom) was examined in the light of the known effects of all three toxins on voltage-dependent K+ currents. 2. When measured in Krebs/phosphate buffer, the binding appeared monotonic at low concentrations of radioiodinated beta-bungarotoxin (Kd 0.4 nM; Bmax 0.42 pmol/mg protein); higher concentrations of labelled toxin revealed an additional binding component of lower affinity, but computer analysis of the data failed to provide well-defined estimates of its Kd and Bmax values. 3. Equilibrium binding experiments conducted in imidazole-based buffers yielded distinctly biphasic Scatchard plots; computer analysis of the data revealed two populations of sites [Kd 0.26 (+/- 0.30) nM and 6.14 (+/- 5.68) nM; Bmax 0.16 (+/- 0.20) and 2.65 (+/- 1.21) pmol/mg protein]. 4. In Krebs medium, beta-bungarotoxin was a very weak antagonist of the binding of 125I-labelled dendrotoxin. In imidazole medium, however, the efficacy of the inhibition was markedly increased; analysis of this inhibition showed it to be non-competitive. 5. Dendrotoxin inhibited the binding of radioiodinated beta-bungarotoxin in Krebs medium with high potency, although the interaction was by a complex, non-competitive mechanism. 6. Mast-cell-degranulating peptide inhibited non-competitively the binding of both radiolabelled dendrotoxin and beta-bungarotoxin but with relatively low potency. 7. A speculative schematic model of the dendrotoxin/beta-bungarotoxin/mast-cell-degranulating peptide binding component(s) is proposed. Findings are discussed in terms of the likely involvement of these sites with voltage-dependent K+-channel proteins.

Involvement of neuronal acceptors for dendrotoxin in its convulsive action in rat brain.

Dendrotoxin, a snake-venom polypeptide, is a potent convulsant that facilitates transmitter release apparently by inhibition of voltage-sensitive K+ channels responsible for A-currents. A biologically active 125I-iodinated derivative of this toxin was prepared and used to characterize kinetically homogeneous non-interacting high-affinity acceptors in synaptic membranes from rat cerebral cortex and hippocampus. Binding of radiolabelled toxin from Dendroaspis angusticeps to its membrane acceptor protein was inhibitable by homologous polypeptides from other mamba snakes; most importantly, their rank order of potency was identical with that for their central neurotoxicities in rats, furnishing evidence for involvement of this binding component in the convulsive symptoms observed. Beta-Bungarotoxin, a presynaptically acting neurotoxin whose action on neurotransmitter release at the neuromuscular junction and effects on brain synaptosomes are antagonized by dendrotoxin, was only able to inhibit the binding of the 125I-labelled toxin with low efficacy, although dendrotoxin apparently interacts avidly with the acceptor sites for beta-bungarotoxin. This weak interaction of beta-bungarotoxin with the acceptor was not attributable to its phospholipolytic action. Other neurotoxins and ion-channel antagonists failed to affect the binding of dendrotoxin. The findings presented here, together with recent electrophysiological data, favour the interpretation that dendrotoxin binds to a membrane protein comprising, or closely associated with, this one group of voltage-dependent K+ channels.

Solution structure of human calcitonin gene-related peptide by 1H NMR and distance geometry with restrained molecular dynamics.

The structure of human calcitonin gene-related peptide 1 (hCGRP-1) has been determined by 1H NMR in a mixed-solvent system of 50% trifluoroethanol/50% H2O at pH 3.7 and 27 degrees C. Complete resonance assignment was achieved by using two-dimensional methods. Distance restraints for structure calculations were obtained by semiquantitative analysis of intra- and interresidue nuclear Overhauser effects; in addition, stereospecific or X1 rotamer assignments were obtained for certain side chains. Structures were generated from the distance restraints by distance geometry, followed by refinement using molecular dynamics, and were compared with experimental NH-C alpha H coupling constants and amide hydrogen exchange data. The structure of hCGRP-1 in this solvent comprises an amino-terminal disulfide-bonded loop (residues 2-7) leading into a well-defined alpha-helix between residues 8 and 18; thereafter, the structure is predominantly disordered, although there are indications of a preference for a turn-type conformation between residues 19 and 21. Comparison of spectra for the homologous hCGRP-2 with those of hCGRP-1 indicates that the conformations of these two forms are essentially identical.

Secondary structure and backbone dynamics of human granulocyte colony-stimulating factor in solution.

The secondary structure and backbone dynamics of the cytokine, human granulocyte colony-stimulating factor (hG-CSF) have been determined by heteronuclear nuclear magnetic resonance (NMR) techniques. Virtually complete NH, C alpha H, C beta H 15N, 13C alpha, and 13C beta assignment of the 175-residue recombinant protein, methionyl-[Cys-17-Ser]-hG-CSF, was achieved by use of three-dimensional (3D) heteronuclear 1H-15N and triple-resonance 1H-15N-13C experiments. Spectra recorded at 750 MHz aided the assignment of severely overlapped regions. The structures of G-CSF from several species have recently been determined by X-ray diffraction [Hill, C. P., Osslund, T. D., & Eisenberg, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5167-5171; Lovejoy, B., Cascio, D., & Eisenberg, D. (1993) J. Mol. Biol. 234, 640-653]. Like several cytokines, hG-CSF has a four-helix topology (A-D) with overhand loop connections, but with an additional helical segment (A') identified in the connection between helix A and helix B. The solution-state determination of the secondary structure is based on short- and medium-range NOEs, backbone J-couplings, and NH exchange data and is corroborated by 13C alpha secondary shifts. The helices are defined as follows: A, 10-38; A',44-53; B, 71-91; C, 102-123; D, 143-172. The dynamics of the amide backbone resonances, investigated using 1H-15N heteronuclear NMR, indicate a rigid protein core with some increased mobility in the AB loop and more pronounced mobility in the CD loop.(ABSTRACT TRUNCATED AT 250 WORDS)

Three-dimensional solution structure and 13C nuclear magnetic resonance assignments of the colicin E9 immunity protein Im9.

The 86-amino acid colicin E9 immunity protein (Im9), which inhibits the DNase activity of colicin E9, has been overexpressed in Escherichia coli and isotopically enriched with 15N and 13C. Using the 3D CBCANH and CBCA(CO)NH experiments, we have almost completely assigned the backbone 13C resonances and extended previously reported 15N/1H backbone assignments [Osborne et al. (1994), Biochemistry 33, 12347-12355]. Side chain assignments for almost all residues were made using the 3D 13C HCCH-TOCSY experiment allied to previous 1H assignments. Sixty solution structures of Im9 were determined using the DIANA program on the basis of 1210 distance restraints and 56 dihedral angle restraints. The 30 lowest-energy structures were then subjected to a slow-cooling simulated annealing protocol using XPLOR and the 21 lowest-energy structures, satisfying the geometric restraints chosen for further analysis. The Im9 structure is well-defined except for the termini and two solvent-exposed loops between residues 28-32 and 57-64. The average RMSD about the average structure of residues 4-84 was 0.94 A for all heavy atoms and 0.53 A for backbone C alpha, C = O, and N atoms. The Im9 fold is novel and can be considered a distorted antiparallel four-helix bundle, in which the third helix is rather short, being terminated close to its N-terminal end by a proline at its C-terminus. The structure fits in well with available kinetic and biochemical data concerning the interaction between Im9 and its target DNase. Important residues of Im9 that govern specificity are located on the molecular surface in a region rich in negatively charged groups, consistent with the proposed electrostatically steered association [Wallis et al. (1995a), Biochemistry 34, 13743-13750].

Structure of an SH2 domain of the p85 alpha subunit of phosphatidylinositol-3-OH kinase.

Receptor protein-tyrosine kinases, through phosphorylation of specific tyrosine residues, generate high-affinity binding sites which direct assembly of multienzyme signalling complexes. Many of these signalling proteins, including phospholipase C gamma, GTPase-activating protein and phosphatidylinositol-3-OH kinase, contain src-homology 2 (SH2) domains, which bind with high affinity and specificity to tyrosine-phosphorylated sequences. The critical role played by SH2 domains in signalling has been highlighted by recent studies showing that mutation of specific phosphorylation sites on the platelet-derived growth factor receptor impair its association with phosphatidylinositol-3-OH kinase, preventing growth factor-induced mitogenesis. Here we report the solution structure of an isolated SH2 domain from the 85K regulatory subunit of phosphatidylinositol-3-OH kinase, determined using multidimensional nuclear magnetic resonance spectroscopy. The structure is characterized by a central region of beta-sheet flanked by two alpha-helices, with a highly flexible loop close to functionally important residues previously identified by site-directed mutagenesis.

NMR trial models: experiences with the colicin immunity protein Im7 and the p85alpha C-terminal SH2-peptide complex.

Two cases of successful molecular replacement using NMR trial models are presented. One is the crystal structure of the Escherichia coli colicin immunity protein Im7; the other is a heretofore unreported crystal structure of a specific PDGF receptor-derived peptide complex of the carboxy-terminal SH2 domain from the p85alpha subunit of human phosphatidylinositol 3-OH kinase. In both cases, molecular replacement was non-trivial. Success was achieved using trial models that consisted of an ensemble of NMR structures from which the more flexible portions had been excised. Use of maximum-likelihood refinement proved critical to be able to refine the poor starting models. The challenges typical of the use of NMR trial models in molecular replacement are discussed.

Structure of a specific peptide complex of the carboxy-terminal SH2 domain from the p85 alpha subunit of phosphatidylinositol 3-kinase.

We have determined the solution structure of the C-terminal SH2 domain of the p85 alpha subunit of human phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) in complex with a phosphorylated tyrosine pentapeptide sequence from the platelet-derived growth factor receptor using heteronuclear nuclear magnetic resonance spectroscopy. Overall, the structure is similar to other SH2 domain complexes, but displays different detail interactions within the phosphotyrosine binding site and in the recognition site for the +3 methionine residue of the peptide, the side chain of which inserts into a particularly deep and narrow pocket which is displaced relative to that of other SH2 domains. The contacts made within this +3 pocket provide the structural basis for the strong selection for methionine at this position which characterizes the SH2 domains of PI3-kinase. Comparison with spectral and structural features of the uncomplexed domain shows that the long BG loop becomes less mobile in the presence of the bound peptide. In contrast, extreme resonance broadening encountered for most residues in the beta D', beta E and beta F strands and associated connecting loops of the domain in the absence of peptide persists in the complex, implying conformational averaging in this part of the molecule on a microsecond-to-millisecond time scale.

Botulinum neurotoxin and dendrotoxin as probes for studies on transmitter release.

Acceptors for BoNT have been detected autoradiographically on the terminal membrane of motor nerves at a density of approximately 150/micron2 and shown to mediate toxin internalization, a process deemed essential for its inhibition of transmitter release. DTX, a protein with pronounced central neurotoxicity, was shown to induce convulsive states in hippocampal slices from guinea-pig. Synaptic transmission was facilitated and spontaneous epileptiform activity produced in intact cell populations. Voltage clamp analysis of hippocampal neurones revealed that DTX specifically attenuated a transient voltage-dependent K+ conductance (A-current) and this could account for the excitatory effects observed. Proteinaceous acceptors with high affinity for DTX were identified on brain synaptosomal membranes and found to contain a 65 000 Mr polypeptide. Their location in rat brain regions was established and contrasted with that of the binding sites for beta-bungarotoxin. These findings indicate the usefulness of DTX as a probe for a protein associated with one variety of K+ channel while the larger subunit of BoNT was found to interact with a membraneous component that resides at cholinergic nerve terminals and, hence, is likely to have a unique role.

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin.

Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24-kDa N-terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP-binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3' position of the noviose sugar is substituted by a 5-methyl-2-pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24-kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5'-adenylyl-beta, gamma-imidodiphosphate, and with information on the bound conformation of novobiocin in the p24-novobiocin complex obtained by heteronuclear isotope-filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented.