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.
Peptides. 2011 Mar;32(3):560-7. doi: 10.1016/j.peptides.2010.06.018. Epub 2010 Jun 30.
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.
Ergtoxin 1（ErgTx1）是一种从墨西哥蝎子 Centruroides noxius Hoffmann 的毒液中纯化得到的 42 个氨基酸肽，能够特异性阻断人类钾通道的醚-α--go-go 相关基因家族（hERG）。该肽结合在通道外口的部分重叠位点上，其中 S5-P 连接子的残基起着关键作用。在这里，我们描述了 ErgTx1 基因的定点突变及其在大肠杆菌中的异源表达的结果。重组产物显示了甲硫氨酸在一级结构中第 35 位（Met35）的基本作用。天然氧化的 Met35 使肽与 hERG1 通道的亲和力降低了三个数量级。这一结果非常重要，因为它表明了两种可能的情况：要么 Met35 参与了分子的正确折叠，要么它在与通道的相互作用中发挥直接作用，即构成相互作用表面的一部分。通过制备异源表达的 ErgTx1 基因和含有 35 位丙氨酸的突变体，评估了这两种情况。此外，还对天然和重组肽进行了圆二色性测量。电生理记录和光学测量得到的结构值强烈支持这样一种观点，即 Met35 确实是毒素与通道相互作用表面上的关键残基。
