Structure and function of Cerebratulus lacteus neurotoxin B-IV: tryptophan-30 is critical for function while lysines-18, -19, -29, and -33 are not required.
The Cerebratulus lacteus B-toxins are a family of polypeptide neurotoxins known to bind to crustacean voltage-sensitive sodium channels. We have previously shown that in the most abundant homolog, toxin B-IV, Arg-17 in the N-terminal helix and a positive charge at position 25 in the loop region are essential for function. In this report, we target a tryptophan residue at position 30, as well as lysine residues found in both the N-terminal helix and loop regions by polymerase chain reaction mutagenesis, to determine their contributions to toxin activity. Substitution of Trp-30 with a serine causes a more than 40-fold reduction in specific toxicity, whereas replacement by tyrosine and phenylalanine is well tolerated. The secondary structures of both these muteins are identical to that of the wild-type toxin as determined by circular dichroism spectroscopy. Thermal denaturation experiments also show that their conformational stabilities are intact. These results demonstrate that an aromatic residue at this position is required for toxin function. Charge neutralizing substitutions of Lys-18 and Lys-19 located in the N-terminal helix have very little effect on toxicity, suggesting the nonessentiality of these residues. Similar results are also obtained for the charge neutralizing muteins for Lys-29 and Lys-33 in the loop region. Interestingly, reduction experiments demonstrate that both K29N and W30S are more sensitive to reducing agent than wild-type B-IV, raising the possibility that the loop sequence may modulate toxin stability.
Wen
P H
PH
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524, USA.
Blumenthal
K M
KM
D000818:Animals; D003400:Astacoidea; D001681:Biological Assay; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D008239:Lysine / Q000235:genetics / Q000502:physiology*; D008387:Marine Toxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009498:Neurotoxins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000235:genetics / Q000502:physiology*
C028056:Cerebratulus lacteus toxin B-IV; D008387:Marine Toxins; D009498:Neurotoxins; D014364:Tryptophan; D008239:Lysine