1. Volume 11, Issue 11, Pages 1381-1392 (November 2003)
Structural Basis of the KcsA K+ Channel and Agitoxin2 Pore-Blocking Toxin Interaction by Using the Transferred Cross-Saturation Method 
Koh Takeuchi, Mariko Yokogawa, Tomoki Matsuda, Mariko Sugai, Seiko Kawano, Toshiyuki Kohno, Haruki Nakamura, Hideo Takahashi, Ichio Shimada 
Structure 
Volume 11, Issue 11, Pages (November 2003)
DOI: /j.str

2. Figure 1 Properties of AgTx2 Binding to the KcsA K+ Channel
(A) Effect of ionic strength on the equilibrium association constant of AgTx2 to the R64D mutant of the KcsA K+ channel. The binding constants were determined by BIACORE 2000 analyses, using a running buffer containing 50 mM Tris-HCl (pH 8.0), 50 μM EDTA, 2 mM KCl, 0.1% DDM, and 0.05% NaN3, and various concentrations of NaCl at 25°C. Each point is the average of at least two independent measurements, and the error bars represent the standard deviation (SD).
(B) K+ ion concentration dependence of AgTx2 binding to the KcsA K+ channel. For the wild-type AgTx2, K+ ion concentration dependence of the binding to the wild-type (purple circles) and the R64D mutant (dark blue diamonds) of the KcsA K+ channel was measured. We also measured K+ ion concentration dependence of AgTx2 mutants binding to the R64D mutant of the KcsA K+ channel. The experiments were carried out for the S11A (pink squares), K27M (green triangles), and M29A (light blue Xs) mutants. The binding constants were determined by BIACORE 2000 analyses, using a running buffer containing 50 mM Tris-HCl (pH 8.0), 50 μM EDTA, 0.1% DDM, 0.05% NaN3, and 150 mM K/NaCl at 25°C. In all experiments, the ionic strength of the running buffer was kept constant by the equimolar substitution of Na+ for K+, using NaCl and KCl, respectively. Each point is the average of at least two independent measurements, and the error bars represent the standard deviation (SD).
Structure  , DOI: ( /j.str )

3. Figure 2 Scheme and Results of the TCS Experiment
(A) Schematic representation of the TCS experiment. The saturation of the nonlabeled KcsA K+ channel caused by the irradiation is transferred to the free state of AgTx2, which is labeled with 2H and 15N.
(B) A 1H-15N HSQC spectrum of AgTx2, which was in complex with 20 mol % of the nonlabeled KcsA K+ channel.
(C) Cross-sections and portions of 1H-15N HSQC spectra observed for the labeled AgTx2, which was complexed with 20 mol % of the nonlabeled KcsA K+ channel, without (left) and with (right) irradiation.
Structure  , DOI: ( /j.str )

4. Figure 3
Determination of the KcsA K+ Channel Interface on AgTx2 by the TCS Measurements
(A) Signal intensity ratios in the TCS experiment. The wild-type AgTx2 was complexed with 20 mol % of the wild-type (red bar) and R64D mutant (blue bar) of the KcsA K+ channel. The ratios for the wild-type KcsA are the average of the results from two independent measurements, and those for the R64D mutant are from three independent measurements. The error bars represent the standard deviation (SD). The secondary structures of AgTx2 are represented by arrows for β sheets and by coiled ribbon for the helix in the plot.
(B) Mapping of the residues affected by the irradiation in the TCS experiments. The color gradient from red to white indicates the signal intensity ratio from 0.6 to 1.0. Gray indicates a N-terminal residue and Pro residues. The left and right figures are 180° rotations about the vertical axis relative to each other.
(C) Mutagenic scans of the KcsA K+ channel interface on AgTx2. Each residue is colored according to the effect of the mutation on the binding affinity. The color gradient from yellow to white indicates the decrease of the binding affinity from 100% to 0%. F25A, which increased the binding affinity 4.6-fold, is also colored. The residues that were not mutated are colored gray.
Structure  , DOI: ( /j.str )

5. Figure 4 Docking of AgTx2 on the KcsA K+ Channel
(A) The model of the AgTx2-KcsA K+ channel complex. The model was build by a simulated annealing calculation. AgTx2 (represented as a yellow tube) is docked on the surface of the KcsA K+ channel (a white surface). The side chains of Arg24, Lys27, and Arg31 of AgTx are represented as blue sticks. Asp64 of KcsA subunits and the ion selective filter are represented as red surfaces. These residues and the ion selective filter are included in the energetic coupling in the KcsA-AgTx binding. See text for details.
(B) The complementarity in the AgTx2-KcsA K+ channel binding interface. Hydrophobic, basic, and acidic residues are colored green, blue, and red, respectively. The residues on the KcsA K+ channel binding surface of AgTx2 and its complementary residues on the channel are connected by the dotted lines.
Structure  , DOI: ( /j.str )

6. Figure 5
Conserved Binding Residues in the Pore-Blocking Toxin–K+ Channel Interface
(A) Comparison of the surface profiles among pore-blocking toxins. Hydrophobic, basic, and acidic residues are colored green, blue, and red, respectively. The functionally important residues are labeled, except for ClTx. For reference, the ClTx residues corresponding to Lys27 and Arg24 of AgTx are labeled. The conserved binding surface is composed of the prominent central Lys, two basic or polar residues at the edge of the surface, and a hydrophobic residue aside next to the central Lys (labeled with pink characters).
(B) Sequence alignments of K+ channels. The sequences of the pore-blocking toxin-sensitive K+ channels, Shaker, Kv1.3, and Kv1.6, and the pore-blocking toxin-insensitive K+ channels, Kv2.1 and Kv3.1, are aligned with the sequence of the R64D mutant of the KcsA K+ channel. The residues that are conserved in more than 50% of the channels are colored as defined in (A). Positions complementary to conserved functionally important residues on pore-blocking toxins are boxed.
Structure  , DOI: ( /j.str )