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	Fig. 2. Design of fragment-based screen for allosteric binders on α7-AChBP. (A) Schematic presentation of the location of the agonist binding site in α7-AChBP as well as possible allosteric binding sites. Competitive binders, the agonist lobeline, and the antagonist d-tubocurarine all compete for binding at the agonist binding site, whereas allosteric binders bind at different sites. (B) To distinguish allosteric binders from competitive binders using SPR spectroscopy we perfused each fragment alone (green triangle) or in combination with the competitive antagonist d-tubocurarine (black circle). In the case of an allosteric binder, the response units observed for the mixture of fragment + d-tubocurarine is close to the sum of fragment alone + d-tubocurarine alone (blue dashed line). No competition exists because the fragment and d-tubocurarine bind at distinct sites. (C) In the case of a competitive binder, the response units for the mixture of fragment + d-tubocurarine is lower than the sum of fragment alone + d-tubocurarine alone because both compounds compete for binding at the same site. (D) Example traces for fragment 4, which was identified as one of the allosteric binders in this study.
	Fig. 3. Allosteric binding at the top pocket in α7-AChBP. (A) Fragment 1 reveals an allosteric binding site located at the N-terminal α-helix of α7-AChBP and is termed the top pocket. α7-AChBP is shown in transparent surface and cartoon representation. Fragment 1 is shown as yellow ball and sticks. (B) Detailed view of fragment 1, wedging between the N-terminal helix and the β2–β3 loop. (C) Binding of fragment 1 causes a conformational change of the N-terminal helix, which is noticeable as a 3-à displacement of residue K12 and a change in the rotamer of L10. The blue ribbon represents the fragment-bound conformation, and the red ribbon represent the apo state. (D) Detailed view of the amino acid interactions between fragment 1 and residues of the top pocket. Fragment 1 is shown in ball and sticks. Yellow is carbon, red oxygen, blue nitrogen, bromine magenta. The green mesh is 2Fo − Fc density contoured at 1.5σ and the magenta mesh is anomalous difference density contoured at 15σ. The black dashed triangles indicate Van der Waals interactions.
	Fig. 4. Allosteric binding at the agonist subpocket and vestibule pocket in α7-AChBP. (A) α7-AChBP is shown in transparent surface and cartoon representation. The black dashed circle indicates the location of the agonist subpocket, which is occupied by fragment 1 right below the orthosteric binding site. (B) Detailed view of the overlapping agonist binding site and agonist subpocket. Lobeline and fragment 1 are shown in ball and stick presentation. (C) Detailed view of the amino acid interactions between fragment 1 and residues of the agonist subpocket. The green mesh is 2Fo − Fc density contoured at 1σ and the magenta mesh is anomalous difference density contoured at 5σ. (D) The location of the vestibule pocket is indicated with the black dashed circle. (E) Detailed view of the vestibule pocket, which is occupied by fragment 4. (F) Detailed view of the amino acid interactions between fragment 4 and residues of the vestibule pocket. The green mesh is 2Fo − Fc density contoured at 1σ and the magenta mesh is anomalous difference density contoured at 10σ.
	Fig. 5. Overview of the different allosteric binding sites discovered in α7-AChBP. α7-AChBP is shown in cartoon representation. Lobeline and different allosteric binders are presented as spheres. The orange ligand corresponds to lobeline and occupies the orthosteric binding site. The red ligand occupies the top pocket, the green ligand occupies the agonist subpocket, and the magenta ligand occupies the vestibule pocket.
	Fig. 6. Functional characterization of allosteric binders on the human α7 nAChR. (A) Example traces of the α7 nAChR repetitively activated with a fixed concentration of acetylcholine (1.3 mM). Coapplication of increasing concentrations of fragment 1 causes a progressive decrease in the ACh-activated current. (B) Concentration-inhibition curves constructed from data shown in A. Error bars indicate SD. (C) Effect of each fragment on the concentration-activation curve of acetylcholine. All of the fragments cause a decrease of the maximal current, without any pronounced effect on the EC50 value, which is indicative for an allosteric effect and is consistent with the crystal structures.

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