Solution structures of a highly insecticidal recombinant scorpion alpha-toxin and a mutant with increased activity.
The solution structure of a recombinant active alpha-neurotoxin from Leiurus quinquestriatus hebraeus, Lqh(alpha)IT, was determined by proton two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). This toxin is the most insecticidal among scorpion alpha-neurotoxins and, therefore, serves as a model for clarifying the structural basis for their biological activity and selective toxicity. A set of 29 structures was generated without constraint violations exceeding 0.4 A. These structures had root mean square deviations of 0.49 and 1.00 A with respect to the average structure for backbone atoms and all heavy atoms, respectively. Similarly to other scorpion toxins, the structure of Lqh(alpha)IT consists of an alpha-helix, a three-strand antiparallel beta-sheet, three type I tight turns, a five-residue turn, and a hydrophobic patch that includes tyrosine and tryptophan rings in a "herringbone" arrangement. Positive phi angles were found for Ala50 and Asn11, suggesting their proximity to functionally important regions of the molecule. The sample exhibited conformational heterogeneity over a wide range of experimental conditions, and two conformations were observed for the majority of protein residues. The ratio between these conformations was temperature-dependent, and the rate of their interconversions was estimated to be on the order of 1-5 s(-1) at 308 K. The conformation of the polypeptide backbone of Lqh(alpha)IT is very similar to that of the most active antimammalian scorpion alpha-toxin, AaHII, from Androctonus australis Hector (60% amino acid sequence homology). Yet, several important differences were observed at the 5-residue turn comprising residues Lys8-Cys12, the C-terminal segment, and the mutual disposition of these two regions. 2D NMR studies of the R64H mutant, which is 3 times more toxic than the unmodified Lqh(alpha)IT, demonstrated the importance of the spatial orientation of the last residue side chain for toxicity of Lqh(alpha)IT.
Tugarinov
V
V
Department of Structural Biology, The Weizmann Institute of Science, Rehovot, Israel.
Kustanovich
I
I
Zilberberg
N
N
Gurevitz
M
M
Anglister
J
J
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics* / Q000633:toxicity; D010572:Pest Control, Biological; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000633:toxicity; D012604:Scorpion Venoms / Q000737:chemistry* / Q000235:genetics* / Q000633:toxicity; D012996:Solutions
C065190:LqhalphaIT toxin; D009498:Neurotoxins; D011994:Recombinant Proteins; D012604:Scorpion Venoms; D012996:Solutions; D006639:Histidine; D001120:Arginine