The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider.

A family of potent insecticidal toxins has recently been isolated from the venom of Australian funnel web spiders. Among these is the 37-residue peptide omega-atracotoxin-HV1 (omega-ACTX-HV1) from Hadronyche versuta. We have chemically synthesized and folded omega-ACTX-HV1, shown that it is neurotoxic, ascertained its disulphide bonding pattern, and determined its three-dimensional solution structure using NMR spectroscopy. The structure consists of a solvent-accessible beta-hairpin protruding from a disulphide-bonded globular core comprising four beta-turns. The three intramolecular disulphide bonds from a cystine knot motif similar to that seen in several other neurotoxic peptides. Despite limited sequence identity, omega-ACTX-HV1 displays significant structural homology with the omega-agatoxins and omega-conotoxins, both of which are vertebrate calcium channel antagonists; however, in contrast with these toxins, we show that omega-ACTX-HV1 inhibits insect, but not mammalian, voltage-gated calcium channel currents.

High resolution NMR solution structure of the leucine zipper domain of the c-Jun homodimer.

The solution structure of the c-Jun leucine zipper domain has been determined to high resolution using a new calculation protocol designed to handle highly ambiguous sets of interproton distance restraints. The domain comprises a coiled coil of parallel alpha-helices in which most of the hydrophobic residues are buried at the highly symmetrical dimer interface; this interface extends over 10 helical turns and is the most elongated protein domain solved to date using NMR methods. The backbone fold is very similar to that seen in crystal structures of the GCN4 and Jun-Fos leucine zippers; however, in contrast with these crystal structures, the Jun leucine zipper dimer appears to be devoid of favorable intermolecular electrostatic interactions. A polar asparagine residue, located at the dimer interface, forms the sole point of asymmetry in the structure; furthermore, the side chain of this residue is disordered due to motional averaging. This residue, which is highly conserved in the leucine zipper family of transcription factors, provides a destabilizing influence that is likely to facilitate the rapid exchange of zipper strands in vivo.

Stereospecificity of substrate usage by glyoxalase 1: nuclear magnetic resonance studies of kinetics and hemithioacetal substrate conformation.

The specificity of glyoxalase 1 for the diastereomers of its hemithioacetal substrate [which forms spontaneously between an alpha-keto aldehyde and reduced glutathione (GSH)] was investigated by exploiting the differences between their 1H NMR spectra at pH* 4.4. The 1H NMR spectra of the hemithioacetals of glutathione with phenylglyoxal or methylglyoxal were assigned with the aid of conventional decoupling and two-dimensional NMR spectroscopic techniques. The rate of interconversion of the diastereomers was determined at 30 degrees C from the results of an inversion-transfer technique and found to be 0.30 +/- 0.04 s-1 (+/- sd) in the case of phenylglyoxal and 0.15 +/- 0.02 s-1 in the case of methylglyoxal. Stereopreference of the enzyme was tested by the addition of large amounts of yeast glyoxalase 1 to a reaction mixture; glyoxalase 1 preferentially operated on one diastereomer of the phenylglyoxal hemithioacetal but the diastereomers of methylglyoxal appeared to be operated upon indiscriminately. From computer models of the kinetics of possible reaction schemes, a mechanism involving glyoxalase 1 catalysis of both diastereomers of the hemithioacetals was shown to be the most consistent with the experimental data. Estimates of internuclear distances in the diastereomers, obtained from 2D NMR spectra were used in "dynamical simulated annealing" calculations to generate likely structures of the substrates. Relative ring-current shifts obtained from 1D NMR spectra were used, together with a ring-current shift algorithm, to select structures with compatible conformations. We conclude that the rate of conversion of substrate by the enzyme is dependent upon the overall conformation of the substrate molecule, rather than merely its stereochemical configuration (R or S).

Determination of the structure of symmetric coiled-coil proteins from NMR data: application of the leucine zipper proteins Jun and GCN4.

Previous attempts to determine the solution structures of homodimeric 'leucine zippers' using nuclear magnetic resonance (NMR) spectroscopy have been impeded by the complete symmetry of these coiled-coil molecules, which makes it impossible a priori to distinguish between intra- and intermonomer dipolar connectivities. Consequently, a number of ad hoc approaches have been used in an attempt to derive tertiary solution structures of these molecules from the NMR data. In this paper we present a more rigorous approach for analysing the NMR spectra of symmetric coiled-coil proteins. This analysis is based on calculations of intra- and intermonomer interproton distances in the recently determined crystal structure of the GCN4 leucine zipper [O'Shea, E.K., Klemm, J.D., Kim, P.S. and Alber, T. (1991) Science, 254, 539-543] and in symmetric coiled-coil models of the leucine zippers of GCN4 and the human oncoprotein Jun which we constructed using a dynamic simulated annealing approach. This analysis has enabled the formulation of a set of rules for interpreting the NMR spectra of symmetric coiled-coil proteins and has also led to the prediction of novel dipolar connectivities which we demonstrate in a 2-D NMR spectrum of the homodimeric Jun leucine zipper.