Structural Basis of the KcsA K+ Channel and Agitoxin2 Pore-Blocking Toxin Interaction by Using the Transferred Cross-Saturation Method

Abstract

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.

Identification and Characterization of the Slowly Exchanging pH-dependent Conformational Rearrangement in KcsA

“…Construction of the KcsA K ϩ Channel Expression VectorThe C-terminal deletion construct, ⌬125-160 KcsA K ϩ channel, was generated from the wild type KcsA construct reported previously (24). The wild type KcsA vector contains the gene encoding the KcsA K ϩ channel with optimal codon usage for Escherichia coli.…”

Atypical Membrane-embedded Phosphatidylinositol 3,4-Bisphosphate (PI(3,4)P2)-binding Site on p47 Phox Homology (PX) Domain Revealed by NMR

“…Transferred cross-saturation (TCS) is an NMR method that enables the identification of the residues of protein ligands in close proximity to huge (Ͼ100 kDa) complexes (19 -21). Successful applications of TCS for huge and/or inhomogeneous systems have previously been reported (22)(23)(24)(25)(26)(27)(28)(29).…”

Structural Basis of Efficient Electron Transport between Photosynthetic Membrane Proteins and Plastocyanin in Spinach Revealed Using Nuclear Magnetic Resonance

“…Our group has developed the transferred crosssaturation (TCS) method, which enables the identification of the residues of protein ligands in close proximity to huge (>100 kD) and/or heterogeneous complexes under fast exchange conditions (Nakanishi et al, 2002;Shimada, 2005). The TCS method has been used to investigate the interactions of large proteins (Nakanishi et al, 2002;Impagliazzo and Ubbink, 2004;Yokogawa et al, 2011), membrane proteins (Takeuchi et al, 2003;Yokogawa et al, 2005;Malia and Wagner, 2007;Kofuku et al, 2009;Yoshiura et al, 2010), liposomes (Takeuchi et al, 2004), and insoluble biomolecules (Nishida et al, 2003;Ichikawa et al, 2007).…”