Bengt E. Westerlund

The three-dimensional structure of notexin, a presynaptic neurotoxic phospholipase A2 at 2.0 A resolution.

The three‐dimensional structure of notexin has been solved by molecular replacement methods. The structure has been refined at 2.0 Å resolution to a crystallographic R‐value of 16.5% with good stereo‐chemistry. The core of the protein is very similar to other phospholipase A2s (PLA2s) but several parts of the molecule are distinctly different. The most significant differences from PLA2s from bovine pancreas and rattlesnake occur in the stretches 56–80 and 85–89. Residue 69, which has been shown to be important for phospholipase binding, has a different conformation and different interactions than in other known PLA2s. The Cα positions for residues 86–88 differ by about 6 Å from both the bovine and the rattlesnake enzyme. The crystals contain no Ca2+ ions. Instead, a water molecule occupies the calcium site.

The three-dimensional structures of two toxins from snake venom throw light on the anticoagulant and neurotoxic sites of phospholipase A2.

The three-dimensional structures of the class II anticoagulant phospholipase A2 (PLA2) toxin RVV-VD from the venom of Russells viper, Vipera russelli russelli, and the class I neurotoxic PLA2 Notechis II-5 from the, Australian tiger snake, Notechis scutatus scutatus, were determined to 2.2 A and 3.0 A resolution, respectively. Both enzymes are monomeric and consist of 121 and 119 residues, respectively. A comparison of ten class I/II PLA2 structures showed, among other differences, that the beta-sheet of these enzymes (residues 76-83) is about 90 degrees less twisted in class I than in class II PLA2s. This, along with the insertion of some residues in the region 57-59 in class I enzymes (the elapid loop), could be the main reason for the significant difference in the anticoagulant and (presynaptic) neurotoxic properties between the two classes of PLA2. It seems apparent from sequence and structural comparisons that the toxic site of PLA2 responsible for the strong anticoagulancy of these toxins consists of a negatively charged part, Glu53, together with a positively charged ridge of lysine residues free for intermolecular interactions. These lysines differ between the two classes of PLA2.

Changes to biological activity following acetylation of dendrotoxin I from Dendroaspis polylepis (black mamba).

The potassium channel blocker dendrotoxin I was acetylated with acetic anhydride. Mono-acetyl derivatives of all seven lysine residues (N-terminus blocked) and a di-derivative were isolated by chromatography on the cation-exchanger Bio-Rex 70 and reversed-phase high-performance liquid chromatography. The derivative acetyl-Lys 29 and the di-derivative of Tyr 24 and Lys 28 had more than 1000 times lower affinity than the native toxin as determined by inhibition of the 125I-dendrotoxin binding to synaptosomal membranes from rat brain. Lys 29 is part of the triplet Lys-Lys-Lys (28-30) which also occurs in the homologous alpha-dendrotoxin where the triplet is not in the functional site, as shown by site-directed mutagenesis. Acetylation of Lys 29 may have produced large structural perturbations that inactivated the toxin. Acetylation of Lys 28 alone had little effect, but the toxin became almost inactive when both Lys 28 and Tyr 24 were modified. Ten experiments were conducted under similar conditions, but a derivative of Tyr 24 was obtained only three times. In these cases the toxin apparently had a different structure, with Tyr 24 accessible to the reagent. This may depend on freeze-drying, which can alter the structure of proteins. The third derivative with low activity was acetyl-Lys 5, with affinity decreased 20-fold. Lys 5 has a protruding side-chain that does not interact with any other group in the toxin molecule. Therefore, Lys 5 is probably part of the functional site for dendrotoxins binding to the voltage-dependent K+ channels.

Crystallographic investigation of the dependence of calcium and phosphate ions for notexin

© 1997 Federation of European Biochemical Societies.

The stellar populations in the Magellanic Clouds. An overview

Recent investigations of the stellar populations in the Magellanic Clouds are reviewed in the light of the evolution and the present structure of the Clouds.