Exploration of the Functional Site of a Scorpion α-like Toxin by Site-Directed Mutagenesis

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Abstract
Scorpion α-neurotoxins can be classified into distinct subgroups according to their sequence and pharmacological properties. Using toxicity tests, binding studies, and electrophysiological recordings, BmK M1, a toxin from the Asian scorpion Buthus martensi Karsch, was experimentally identified as an α-like toxin. Being the first α-like toxin available in a recombinant form, BmK M1 was then modified by site-directed mutagenesis for investigation of the molecular basis of its activity. The results suggested a functional site which protrudes from the molecular scaffold as a unique tertiary arrangement, constituted by the five-residue reverse turn 8−12 and the C-terminal segment. The C-terminal basic residues Lys62 and His64 together with Lys8 in the turn, which are critical for the bioactivities, may directly interact with the receptor site on the sodium channel. Residues Asn11 and Arg58, indispensable for the activities, are mainly responsible for stabilizing the distinct conformation of the putative bioactive site. Among others, His10 and His64 seem to be involved in the preference of BmK M1 for phylogenetically distinct target sites. The comparison of BmK M1 with Aah2 (classical α-toxin) and LqhαIT (α-insect toxin) showed that the specific orientation of the C-terminus mediated by the reverse turn might be relevant to the preference of α-toxin subgroups for phylogenetically distinct yet closely related receptor sites. The Y5G mutation indicated the “conserved hydrophobic surface” might be structurally important for stabilizing the β-sheet in the α/β-scaffold. The observations in this work shed light on the nature and roles of the residues possibly involved in the biological activity of a scorpion α-like toxin.