Structural basis of the KcsA K⁺ channel and agitoxin2 pore-blocking toxin interaction by using the transferred cross-saturation method

We have determined the binding site on agitoxin2 (AgTx2) to the KcsA K ⁺ channel by a transferred cross-saturation (TCS) experiment. The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K⁺ channel. Based on properties of the AgTx2 binding site with the KcsA K⁺ channel, we present a surface motif that is observed in pore-blocking toxins affecting the K⁺ channel. Furthermore, we also explain the structural basis of the specificity of the K⁺ channel to the toxins. The TCS method utilized here is applicable not only for the channels, which are complexed with other inhibitors, but also with a variety of regulatory molecules, and provides important information about their interface in solution.