Recombinant expression of the toxic peptide ErgTx1 and role of Met35 on its stability and function.
Ergtoxin 1 (ErgTx1) is a 42 amino acid peptide purified from the venom of the Mexican scorpion Centruroides noxius Hoffmann, capable of blocking specifically human potassium channels of the ether-á-go-go-related gene family (hERG). This peptide binds to a partially overlapping site on the channel outer mouth, in which residues of the S5-P linker are critically involved. Here we describe results of site directed mutagenesis of the ErgTx1 gene and its heterologous expression in Escherichia coli. The recombinant products show the fundamental role played by methionine in position 35 (Met35) of the primary structure. Naturally oxidized Met35 decreases by three orders of magnitude the affinity of the peptide for the hERG1 channels. This result is quite relevant, because it shows two possible situations: either Met35 is involved in the proper folding of the molecule or it plays a direct role in the interaction with the channel, i.e., constitutes part of the interacting surfaces. These two situations were evaluated by preparing heterologously expressed ErgTx1 gene and a mutant containing alanine in position 35. Additionally circular dichroism measurements of both native and recombinant peptides were performed. The electrophysiological recordings and the structural values obtained by optical measurements, strongly support the idea that Met35 is indeed a key residue on the interacting surfaces of the toxin with the channels.
Jimenez-Vargas
J M
JM
Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Av. Universidad, 2001, P. O. Box 501-3, Cuernavaca 62210, Mexico. jimenez@ibt.unam.mx
Restano-Cassulini
R
R
Quintero-Hernández
V
V
Gurrola
G B
GB
Possani
L D
LD
D000818:Animals; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D004594:Electrophysiology; D005260:Female; D020128:Inhibitory Concentration 50; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D012604:Scorpion Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D012604:Scorpion Venoms; C118951:ergotoxin, Centruroides noxius