Transfer of a beta-hairpin from the functional site of snake curaremimetic toxins to the alpha/beta scaffold of scorpion toxins: three-dimensional solution structure of the chimeric protein.
The alpha/beta scorpion fold is shared by scorpion toxins, insect defensins, and plant thionins. This small and functionally versatile template contains an alpha-helix and a triple beta-sheet linked by three disulfide bridges. With the view to introduce novel functional centers within this fold, we replaced the sequence (the cysteines and glycines excepted) of the original beta-hairpin of a scorpion toxin by the sequence of a beta-hairpin that forms part of the site by which snake neurotoxins bind to nicotinic acetylcholine receptors (AcChOR). The resulting chimeric protein, synthesized by chemical means, binds to AcChOR, though with a lower affinity than the snake toxins [Drakopoulou; E., Zinn-Justin, S., Guenneugues, M., Gilquin, B., Ménez, A., & Vita, C. (1996) J. Biol. Chem. 271, 11979-11987]. The work described in this paper is an attempt to clarify the structural consequences associated with the transfer of the beta-hairpin. We report the determination of the three-dimensional solution structure of the chimeric protein by proton NMR spectroscopy and molecular dynamics calculations. Comparison of the structure of the chimera with those of the scorpion alpha/beta toxin and of the snake neurotoxin shows that (i) the new protein folds as an alpha/beta motif and (ii) the beta-hairpins of the chimera and of the curaremimetic toxin adopt a similar conformation. A closer inspection of the differences between the structures of the original and transferred beta-hairpins allows rationalization of the biological properties of the chimera.
Zinn-Justin
S
S
CEA, Département d'Ingénierie et d'Etude des Protéines, Gif-sur-Yvette, France. szinn@balthazar.saclay.cea.fr
Guenneugues
M
M
Drakopoulou
E
E
Gilquin
B
B
Vita
C
C
Ménez
A
A
D000595:Amino Acid Sequence; D018999:Charybdotoxin / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012604:Scorpion Venoms / Q000737:chemistry*; D012910:Snake Venoms / Q000737:chemistry*
D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D012910:Snake Venoms; D018999:Charybdotoxin