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	Figure 1. Isolation and characterization of α-conotoxin TxIA. (A) Ls-AChBP screening for α-conotoxins in venoms of 30 species of Australian cone snails. (B) LC-MS profile of the crude venom of C. textile. TxIA was isolated as a minor component shown in the extracted ions for this peptide. Inset shows a shell of C. textile. (C) Saturation binding experiments revealed a competitive interaction of TxIA with Ls-AChBP. When an ∼IC50 concentration of TxIA was added, the Kd of 125I-Bgt shifted from 3.5 to 40 nM, whereas the Bmax remained unaffected. (D) Displacement of 125I-Bgt from Ls-AChBP by α-conotoxins TxIA, PnIA and analogs (plot with all fixed to 100% in curve fit). Data in (A), (C) and (D) represent the mean±s.e.m. of duplicate data obtained in three separate experiments.
	Figure 2. Antagonist activity of α-conotoxin TxIA and analogs at oocyte-expressed rat α3β2 (A) and α7 (B) nAChRs. Oocytes were clamped at −70 mV and 100 μM ACh (α3β2) or nicotine (α7) were applied for 2 s in 4 min intervals. Toxins were applied for 3 min. Data are represented as the mean±s.e.m. of at least four oocytes.
	Figure 3. (A) Crystal structure of Ac-AChBP in complex with TxIA(A10L). The model is shown along the five-fold symmetry axis. α-Conotoxins are shown in red. (B) Superposition of the subunit interface from Ac-AChBP in complex with TxIA(A10L) and PnIA(A10L D14K). The principal face of the binding site is shown in shades of yellow, the complementary face in shades of blue. The superposition illustrates the different orientation of the conotoxin backbone in the binding pocket. PnIA(A10L D14K) is shown in blue, TxIA(A10L) in red. (C) Comparison of the different backbone orientations observed in co-crystal structures of Ac-AChBP with different α-conotoxins. TxIA(A10L), shown in red, is tilted by a 20° rotation around Pro7 with respect to PnIA(A10L D14K), shown in blue. The backbone orientation of α-conotoxin ImI, shown in magenta, is very similar to PnIA(A10L D14K) even though it is much shorter and forms a different network of interactions. Lines represent disulfide bridges. Detailed view of the molecular interactions that results in the different backbone orientations of (D) TxIA, (E) PhIA(A10L D14K) and (F) ImI within the binding site. The principal face of the binding site is shown in yellow, the complementary face in blue. The conotoxins are colored according to the color scheme in (C). Dashed lines represent hydrogen bonds or electrostatic interactions.
	Figure 4. Surface representation of the three α-conotoxins that have been co-crystallized with Ac-AChBP. The surface of the α-conotoxin facing the principal binding site is shown in (A), the complementary binding site in (B). Ac-AChBP is shown in a transparent view for clarity. The principal subunit (+) of Ac-AChBP is shown in yellow, the complementary subunit (−) in blue.
	Figure 5. Functional contribution of the electrostatic interaction between Arg5-TxIA and an Asp residue of the principal binding subunit of Ac-AChBP and different nAChR subtypes. (A) Ki estimates from binding assays on wild-type Ac-AChBP, D195A and D195N mutants demonstrating the loss of affinity for Arg5-containing conotoxins. (B) Surface plasmon resonance experiments on wild-type Ac-AChBP (red traces) and D195A mutant (blue traces) for TxIA analogs and PnIA(A10L D14K). (C) Effect of the Asp-mutations on the potency of TxIA and analogs to inhibit α7 and α3β2 nAChR current. (D, E) Homology models for the interaction of TxIA(A10L) with α3β2 and α7 nAChRs. The α3β2 model also shows the Arg–Asp interaction observed in the co-crystal structure.

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