Renée Ménez

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity.

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle α1βγδ-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 Å by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snakes diet.

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering.

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the “three‐finger” fold and the short α/β scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short α/β scorpion fold is highly compact, whereas the “three‐finger” fold is a β structure presenting large flexible loops. First, the crystal structure of the snake toxin α was solved at 1.8‐Å resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin α and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X‐ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X‐ray structure are in agreement with the experimental NMR and X‐ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin α. The regions submitted to rare motions during the simulations are those that exhibit millisecond time‐scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large‐scale motions should be those tolerant to large insertions or deletions.

Engineering of three-finger fold toxins creates ligands with original pharmacological profiles for muscarinic and adrenergic receptors.

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.

Three-dimensional crystal structure of recombinant erabutoxin a at 2.0 Å resolution

Recombinant erabutoxin a (Ear) has been crystallized by vapour diffusion in hanging drops. The crystals belong to space group P212121 with cell dimensions a = 55.8 Å, b = 53.4 Å, c = 40.8 Å. Diffraction data have been recorded on a FAST detector up to 2.0 Å. The atomic crystal structure of Ear has been determined by initial refinement of the structure of the isotoxin erabutoxin b (Eb) the crystals of which were grown under identical conditions. The R‐factor was 23% at 2.0 Å resolution. The secondary and tertiary structures of Ear are shown to be identical with that of wild‐type Eb, within the experimental error.

Preliminary X-ray analysis of crystals of fasciculin 1, a potent acetylcholinesterase inhibitor from green mamba venom*

Fasciculin 1 from Dendroaspis angusticeps has been crystallized by vapour diffusion, in sodium acetate using sodium thiocyanate as precipitant. Tetragonal crystals (space group P4(1)2(1)2 or P4(3)2(1)2) diffract to 1.8 A resolution. The unit cell parameters are a = 40.4 A and c = 81.1 A. We estimated the presence of one molecule in the asymmetric unit.