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Crystal structure of a Cbtx–AChBP complex reveals essential interactions between snake α-neurotoxins and nicotinic receptors

2005; Springer Nature; Volume: 24; Issue: 8 Linguagem: Inglês

10.1038/sj.emboj.7600620

1460-2075

Yves Bourne, Todd T. Talley, Scott B. Hansen, Palmer Taylor, P. Marchot,

Ion channel regulation and function

Article24 March 2005free access Crystal structure of a Cbtx–AChBP complex reveals essential interactions between snake α-neurotoxins and nicotinic receptors Yves Bourne Corresponding Author Yves Bourne Architecture et Fonction des Macromolécules Biologiques, CNRS UMR-6098, Marseille, France Search for more papers by this author Todd T Talley Todd T Talley Department of Pharmacology 0636, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Scott B Hansen Scott B Hansen Department of Pharmacology 0636, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Palmer Taylor Palmer Taylor Department of Pharmacology 0636, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Pascale Marchot Corresponding Author Pascale Marchot Ingénierie des Protéines, CNRS FRE-2738, Institut Fédératif de Recherche Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur Nord, Marseille, France Search for more papers by this author Yves Bourne Corresponding Author Yves Bourne Architecture et Fonction des Macromolécules Biologiques, CNRS UMR-6098, Marseille, France Search for more papers by this author Todd T Talley Todd T Talley Department of Pharmacology 0636, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Scott B Hansen Scott B Hansen Department of Pharmacology 0636, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Palmer Taylor Palmer Taylor Department of Pharmacology 0636, University of California at San Diego, La Jolla, CA, USA Search for more papers by this author Pascale Marchot Corresponding Author Pascale Marchot Ingénierie des Protéines, CNRS FRE-2738, Institut Fédératif de Recherche Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur Nord, Marseille, France Search for more papers by this author Author Information Yves Bourne 1, Todd T Talley2, Scott B Hansen2, Palmer Taylor2 and Pascale Marchot 3 1Architecture et Fonction des Macromolécules Biologiques, CNRS UMR-6098, Marseille, France 2Department of Pharmacology 0636, University of California at San Diego, La Jolla, CA, USA 3Ingénierie des Protéines, CNRS FRE-2738, Institut Fédératif de Recherche Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur Nord, Marseille, France *Corresponding authors: Architecture et Fonction des Macromolécules Biologiques, CNRS UMR-6098, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. E-mail: [email protected]é de la Méditerranée, Faculté de Médecine Secteur Nord, Ingénierie des Protéines, Blvd Pierre Dramard, 13916 Marseille Cedex 20, France. Tel.: +33 4 91 69 89 08; Fax: +33 4 91 65 75 95; E-mail: [email protected] The EMBO Journal (2005)24:1512-1522https://doi.org/10.1038/sj.emboj.7600620 Correction(s) for this article Crystal structure of a Cbtx–AChBP complex reveals essential interactions between snake α-neurotoxins and nicotinic receptors11 January 2006 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The crystal structure of the snake long α-neurotoxin, α-cobratoxin, bound to the pentameric acetylcholine-binding protein (AChBP) from Lymnaea stagnalis, was solved from good quality density maps despite a 4.2 Å overall resolution. The structure unambiguously reveals the positions and orientations of all five three-fingered toxin molecules inserted at the AChBP subunit interfaces and the conformational changes associated with toxin binding. AChBP loops C and F that border the ligand-binding pocket move markedly from their original positions to wrap around the tips of the toxin first and second fingers and part of its C-terminus, while rearrangements also occur in the toxin fingers. At the interface of the complex, major interactions involve aromatic and aliphatic side chains within the AChBP binding pocket and, at the buried tip of the toxin second finger, conserved Phe and Arg residues that partially mimic a bound agonist molecule. Hence this structure, in revealing a distinctive and unpredicted conformation of the toxin-bound AChBP molecule, provides a lead template resembling a resting state conformation of the nicotinic receptor and for understanding selectivity of curaremimetic α-neurotoxins for the various receptor species. Introduction The soluble acetylcholine-binding protein (AChBP) from the freshwater snail Lymnaea stagnalis is a structural homolog of the extracellular ligand-binding domain of muscle-type and neuronal nicotinic acetylcholine receptors (nAChRs) (Brejc et al, 2001; Smit et al, 2001) and other pentameric receptors of the Cys-loop members in the ligand gated ionic channel (LGIC) superfamily (Le Novère and Changèux, 1999). AChBP shows ∼24% sequence identity with the neuronal α7 nAChR (Figure 1A) and assembles as a homopentamer that binds the classical alkaloid agonists and antagonists with dissociation constants characteristic of the nAChRs (Taylor et al, 2000; Changeux and Edelstein, 2001; Grutter and Changeux, 2001; Karlin, 2002). Figure 1.Sequences and numbering of AChBP and Cbtx. (A) Sequence of the L. stagnalis AChBP subunit, aligned with those of the A. californica AChBP subunit (Hansen et al, 2004), of various subunits from human (Hu) and T. californica (Tc) nAChR subtypes (LGIC database), and of the 13-mer high-affinity peptide (Harel et al, 2001). The loop C tip is indicated by a bar above the alignment. Tip up and down triangles respectively denote AChBP residues from the principal and complementary faces of the subunit interface that interact with Cbtx. (B) Structural alignment of the Cbtx sequence with those of the long α-neurotoxins Bgtx and LSIII (bind muscle-type and neuronal nAChRs), of κ-Bgtx (binds neuronal nAChRs), and of the short α-neurotoxins erabutoxin (Ebtx) and NmmI (bind muscle-type nAChRs). The fifth disulfide bridge present in loop II in the long α-neurotoxins and the κ-neurotoxins is indicated by a bar above the alignment. Tip up and down triangles respectively denote Cbtx residues that interact with the AChBP principal and complementary faces of the subunit interface; filled circles denote residues interacting with both faces. A full-color version of this figure is available at The EMBO Journal Online (Supplementary Figure 1). Download figure Download PowerPoint AChBP also associates with the postsynaptic, curaremimetic α-neurotoxins from snake venom in a manner similar to the skeletal muscle α12βγδ and neuronal α7 nAChRs (Smit et al, 2001; Hansen et al, 2002, 2004). These three-fingered toxins, exemplified by the long α-neurotoxin, α-bungarotoxin (Bgtx), a potent antagonist of the muscle receptor (Chang and Lee, 1963; Changeux et al, 1970), have defined molecular probes and pharmacological tools to investigate the structural and functional biology of the nAChRs. Since then, the long α-cobratoxin (Cbtx) and toxin LSIII and the short erabutoxin, toxin-α, and toxin NmmI (Figure 1B) have also been used as selective ligands for studying the nAChRs (Endo and Tamiya, 1991; Fruchart-Gaillard et al, 2002; Taylor et al, 2002). These peptidic toxins appear unique among the ligands because of their distinctive binding kinetics and remarkably high affinity and selectivity for the various nAChR subtypes; they may also provide lead compounds for the design of clinically useful drugs (Taylor et al, 2002; Tsetlin and Hucho, 2004). Hence, understanding their mode of interaction and defining the interface of the toxin–receptor complexes have been areas of substantial interest in neurobiology for four decades (Nirthanan and Gwee, 2004). The availability of the primary AChBP structure (Brejc et al, 2001), buttressed by a wealth of pharmacology, mutagenesis, and chemical modification data accumulated over years (Taylor et al, 2000; Karlin, 2002), has prompted an emergence of theoretical models of α-neurotoxin–nAChR complexes, designed from structures of long α-neurotoxins bound to synthetic peptides derived from the α1 subunit sequence (Harel et al, 2001; Moise et al, 2002; Samson et al, 2002; Zeng and Hawrot, 2002) and from mutational analysis of pairwise interactions between Cbtx and an α7 receptor (Fruchart-Gaillard et al, 2002). However, these models, which differ from one another, have been restricted to the AChBP conformation observed in the initial template and only address limited components of the overall toxin–receptor interface. Early on, the muscle-type nAChR was shown to undergo conformational transitions between resting, desensitized, and open channel states, each with a distinctive affinity for acetylcholine (cf Monod et al, 1965; Changeux and Edelstein, 2001). In turn, α-neurotoxins have been proposed to stabilize the resting state of the nAChR (Moore and McCarthy, 1995). The AChBP conformation observed in the initial structure was thought to reflect primarily the desensitized state of the nAChR, while occurrence of a resting state would require conformational rearrangements in the quaternary structure of the protein (Grutter and Changeux, 2001). Tryptophan fluorescence quenching data have suggested that Bgtx binding induces a unique conformational state of AChBP (Hansen et al, 2002) and that acetylcholine binding induces conformational changes leading to an occluded binding site (Gao et al, 2004; Hibbs et al, 2004). Moreover, channel gating was achieved with a concomitant reduction in agonist affinity from expression of a chimeric cDNA encoding a suitably modified AChBP connected to the transmembrane spans of the 5HT-3 receptor, also a member of the pentameric LGIC family (Bouzat et al, 2004). The ligand-binding and channel gating data are consistent with AChBP retaining the capacity to undergo conformational transitions between states with distinctive agonist affinities and channel conductance properties. We have solved the crystal structure of the complex formed between the long α-neurotoxin, Cbtx, and Lymnaea AChBP (Table I). This structure, with five toxin molecules bound to the pentameric receptor, reveals conformational changes at the AChBP subunit interfaces that are much larger than those observed for AChBP bound with small nicotinic agonists (Celie et al, 2004). Hence, it establishes how α-neurotoxins bind to the nAChRs and distinguishes between the various models of complexes designed from the primary AChBP structure. Following the lead structure of a related three-fingered toxin, fasciculin Fas2, bound to acetylcholinesterase (AChE) (Bourne et al, 1995), this structure describes a second distinctive, high-affinity complex between a snake three-fingered toxin and a synaptic acetylcholine-recognition protein. Table 1. Data collection and refinement statistics Data collectiona Beamline (ESRF) ID14-EH4 Wavelength (Å) 0.975 Space group C2221 Cell dimensions (Å) a=162.6, b=313.4, c=106.5 Resolution range (Å) 30–4.2 Total observations 60 284 Unique reflections 19 232 Multiplicity 3.1 (2.5) Completeness (%) 95.1 (86.8) I/σ(I) 6.7 (2.7) Rsymb 16.0 (36.6) B from Wilson plot (Å2) 41.1 Refinementc R-factor/R-free (%) 33.1 (35.2)/37.8 (47.1) Average B-factor (Å2) 39.8 R.m.s.d.d Bonds (Å)/angles (deg) 0.012/1.4 Chiral volume (Å3) 0.081 Validation Map correlation Main/side chainse 0.77 (0.77)/0.68 (0.74) Ramachandran plotf Outliers (%) AChBP: 5.1 Cbtx: 9.1 Structure Z-scoresg Second-generation packing quality −2.889 Ramachandran plot appearance −2.112 Chi-1/chi-2 rotamer normality −2.130 Backbone conformation 0.004 a Values in parentheses are those for the 4.31–4.2 Å resolution shell. b Rsym=∑hkl∑i∣Ii(hkl)−∣〈Ihkl〉∣∣/∑hkl∑iIi(hkl), where I is an individual reflection measurement and 〈I〉 the mean intensity for symmetry-related reflections. c R-factor=∑hkl∣∣Fo∣−∣Fc∣∣/∑hkl∣Fo∣, where Fo and Fc are observed and calculated structure factors, respectively. R-free is calculated for 5% of randomly selected reflections excluded from refinement. d Root mean square deviation from ideal values, according to Engh and Huber (1991). e According to Branden and Jones (1990). Values in parentheses are those for the interacting residues listed in Table II. f According to Kleywegt and Jones (1996). g According to WHATIF (Hooft et al, 1996). Results and discussion Preparation and analysis of the Cbtx–AChBP complex in solution To optimize occupancy of all five binding sites on AChBP, yet minimize excess of unbound toxin that may preclude crystallization, concentration ratios were carefully adjusted in forming the Cbtx–AChBP complex (Figure 2). As expected from the relative masses and net charges of Cbtx and the glycosylated AChBP subunit, and from the nanomolar dissociation constant of the complex (Hansen et al, 2002), the bound Cbtx molecules were found to reduce the mobility of AChBP in native-PAGE. Moreover, titration of the fractional occupancies of the five AChBP subunit interfaces revealed a linear progression in the appearance of all four intermediate complexes before saturation at stoichiometry is achieved. The same reductions in AChBP mobility and intermediate complexes were observed when Bgtx, which also binds AChBP with high affinity (Hansen et al, 2002), was used. By contrast, the cationic short α-neurotoxin of lower affinity, NmmI, yielded a broadened band suggestive of fractional occupation of the sites (not shown). Figure 2.Analysis of Cbtx–AChBP complex formation. Complex formation and stoichiometry were analyzed by native-PAGE with migration toward the anode (bottom). The progressive shift toward the cathode in the positions of the intermediate complexes (molar ratios of 0.3, 0.6, 0.9, 1.2, and 1.5 Cbtx per binding site; lanes 2–6) relative to unliganded AChBP (5 × 30 kDa; pI 5.0; lane 1) denotes increasing fractional occupancies (lanes 2–4) and then full occupancy (lanes 5 and 6) of the AChBP pentamer by Cbtx (8 kDa; pI 8.6). The unbound Cbtx in excess (lanes 5 and 6) migrates toward the cathode and off the gel. Download figure Download PowerPoint Gel filtration of the immunoaffinity-purified AChBP followed by electrophoretic analyses indicated the presence of monomers and dimers of the pentameric molecule, both yielding the same single band in SDS–PAGE and native-PAGE (not shown). The Cbtx–AChBP complexes prepared from the purified monomers and dimers were both found to elute as dimers of pentameric complexes. This suggests that distinctive dimers of the complex may assemble by end on stacking of the pentameric rings or lateral stacking via the bound toxins. Crystal packing and quality of the structure The structure of the Cbtx–AChBP complex was solved by molecular replacement and carefully refined at 4.2 Å resolution (Table I). In the crystal, despite the large solvent channel that biases the overall resolution (cf Materials and methods), two packing interfaces for the pentameric complexes result in very well-ordered bound toxin molecules and binding interfaces (Figures 3 and 4). At the first packing interface, where coaligned dimers of pentamers assemble tail-to-tail as found in the Hepes-bound AChBP crystal (Brejc et al, 2001), the clustered five AChBP C-termini diverge laterally at the protonated Arg206 residues (Figure 1A) but the uncharged 6xHis residues may contribute to the dimer formation (not shown). At the second packing interface, the external, third β-strand in a bound Cbtx molecule closely interacts with the AChBP-exposed antiparallel segment in loop β8–β9 from a symmetry-related pentameric complex (Figure 3C). This interaction results in a Cbtx-mediated lateral association of complexes, where the entrances to the subunit interfaces in two distinct pentamers are separated by 20–24 Å, a distance shorter than that between two ligand-binding sites within a pentamer. Incidentally, this interaction mimics the dimeric assemblies of reversely oriented three-fingered toxin molecules linked by their third β-strands and C-terminal regions observed in structures of α- and κ-neurotoxins (Love and Stroud, 1986; Betzel et al, 1991; Oswald et al, 1991; Dewan et al, 1994). These two arrangements are fully consistent with electron microscopy images of a crystalline Bgtx–nAChR complex (Paas et al, 2003) and with the distinctive dimer assemblies observed by gel filtration (cf above). A third mode of association between complexes in the crystal involves the second β-strand and C-terminal segment of Cbtx. Figure 3.Overall view of the Cbtx–AChBP complex. The pentameric complex is viewed along (A) and perpendicular (B) to the AChBP five-fold axis. AChBP subunits A and B, which respectively contribute to the principal and the complementary faces of the interface, are displayed in yellow and blue; the Cbtx molecule bound at this subunit interface is in purple. (C) Cbtx-mediated assembly of pentameric complexes in the crystal. The AChBP segment Thr155–Ser159 in loop β8–β9 and the Cbtx antiparallel strand β3 (Asp53–Cys57) are shown in green. The crystalline homodimeric assembly of two reversely oriented Cbtx molecules (purple) through their antiparallel strand β3 (green) (structure 2CTX; Betzel et al, 1991) is shown on the right. Download figure Download PowerPoint Figure 4.Quality of the Cbtx–AChBP complex structure. (A–C) Views of the 4.2 Å averaged electron density maps for (A) the bound Cbtx molecule, viewed down to the concave face (purple Cα; cyan map contoured at 1σ) and its five disulfide bridges (orange Cα and green bonds; blue map contoured at 2.5σ); (B) the newly open conformation of loop C (yellow Cα; cyan map contoured at 1σ) compared with its conformation in Hepes-bound AChBP (orange Cα) (structure 1I9B; Brejc et al, 2001); (C) the complex interface (stereo view; cyan map contoured at 1σ); Cbtx residues Phe29 and Arg33 partially mimic the nicotine molecule (purple; transparent molecular surface) superimposed as bound to AChBP (structure 1UW6; Celie et al, 2004). Download figure Download PowerPoint As a result of the full ligand occupation and the tight packing interactions, the quality of the density maps (Figure 4) along with availability of high-resolution structures for each of the two complex partners permits unambiguous positioning of all secondary structure elements in the Cbtx–AChBP complex and most of the side chains at the binding interfaces. Hence, structural comparison of AChBP bound to the antagonist Cbtx with AChBP bound to the agonists, nicotine and carbamoylcholine (Celie et al, 2004), reveals the unique conformational changes induced by the α-neurotoxin (Figures 4, 5 and 6). Figure 5.Conformational changes in AChBP associated with Cbtx binding. (A) Superimposition of AChBP subunits A (yellow) and B (blue) in the Cbtx complex with those in the Hepes-bound AChBP structure (gray). AChBP loops C and F, which undergo conformational changes upon Cbtx binding, are respectively displayed in orange and green for the toxin complex and in dark blue and magenta for the Hepes-bound conformation. (B) Close-up view of the conformational changes (indicated by the arrows) within the ligand-binding site, with loop C on left and loop F on right (same colors as in panel A). The likely hydrogen-bonding network is based on interatomic distances. A nicotine molecule (purple; transparent molecular surface) is superimposed as bound to AChBP. Download figure Download PowerPoint Figure 6.The Cbtx–AChBP complex interface. (A) Close-up view of the bound Cbtx molecule. The Cbtx residues (black labels) that interact with the principal and complementary faces of the AChBP subunit interface are in yellow and cyan/green, respectively. The AChBP residues in the complementary face of the interface are in white (blue labels). (B) Structural comparison of (left) the Cbtx-bound loop Ser182–Glu193 of AChBP and (right) the Bgtx-bound 13-mer peptide (structure 1HC9; Harel et al, 2001). The molecular surfaces of the complex interfaces are shown in transparency. Download figure Download PowerPoint Overall view of the Cbtx–AChBP complex The three-fingered Cbtx molecule consists of two antiparallel β-sheets with a central three-stranded β-sheet formed by residues Cys20–Trp25, Arg36–Gly40, and Asp53–Cys57 (Betzel et al, 1991) (Figures 1B and 4A). The Cbtx three loops, loops I, II, and III, that emerge from the dense core containing four disulfide bridges, form a slightly concave disk elongated in the direction of the long loop II that bears the Trp25-Cys-Asp-Ala-Phe-Cys-Ser31 sequence and fifth disulfide bond characteristic of the long α-neurotoxins. In the complex, Cbtx loop II inserts deeply into the ligand-binding pocket, located at the interface between two AChBP subunits (referred to as A for the principal face where loop C resides and B for the complementary face where loop F resides) (Figures 3 and 5). AChBP subunits A and B contribute mean values of 700 and 425 Å2, respectively, to the Cbtx–AChBP interfacial area buried to a 1.6 Å probe radius. An interfacial area in the 1125 Å2 range represents 20–25% of the total accessible surface area of Cbtx and comprises 18 residues of Cbtx, of which four are positively charged (Table II). The buried surface area and number of residues involved are similar to those calculated for the Fas2–AChE complex interface (Bourne et al, 1995). The Cbtx molecular axis, defined by the direction of the central three-stranded antiparallel β-sheet, lies at an ∼45° angle relative to the median axis of the AChBP ring and is near-perpendicular to the cylinder wall (Figure 3). As a result, the center of gravity of Cbtx is located exactly at a midposition in the 62 Å high AChBP cylinder, and the toxin disulfide core resides closer to the ‘membrane’ side than the apical side of loop C. Protrusion of the five bound toxins at the outer perimeter of the AChBP pentamer significantly extends, by 50 Å, the radial dimension of the cylinder. The resulting total diameter of 130 Å for the pentameric Cbtx–AChBP complex is consistent with the size of spherical particules observed by electron microscopy from nanocrystals of a Bgtx–nAChR complex (Paas et al, 2003). Table 2. Intermolecular interactionsa Cbtxb AChBP subunit interface Principal face (subunit A) Complementary face (subunit B) Loop I Thr6 Thr184 Pro7 (Ala7) Ser182, Thr184, Glu190, Ala191 Ile9 Glu190 Loop II Trp25 Glu163 Cys26–Cys30 Glu157, Asp160 Asp27 Tyr185 Ala28 Lys34, Tyr164 Phe29 Tyr185, Tyr192 Trp53 Ser31 Gln55, Thr155 Ile32 (Ser35) Gln55,Leu112, Met114 Arg33 Thr144, Cys187, Tyr192 Arg104 Gly34 Ser186 Lys35 Ser186 Arg36 (Val39) Tyr185 Val37 Thr184, Ser186 C-terminus Phe65 (His68) Thr184, Cys187, Pro189 Arg68 (Gln71) Ser186 a Within a 4.5 Å distance between atoms from each partner in the complex. b Residue substitutions in Bgtx are indicated in parentheses. c Indicated in bold are those residues in Cbtx and in the α7 receptor (AChBP numbering) whose mutations cause an affinity decrease of more than five- and seven-fold, respectively (Fruchart-Gaillard et al, 2002). Superimposition of the Cbtx-bound AChBP pentamer with the Hepes- and agonist-bound pentamers (Brejc et al, 2001; Celie et al, 2004) unambiguously reveals that toxin binding is associated with major positional changes of AChBP loops C and F that border the ligand-binding pocket (Figures 4 and 5). These two loops, which respectively belong to the principal and complementary sides of the subunit interface, are markedly displaced, by up to 10 Å, to uncap the pocket. However, Cbtx binding does not significantly alter the relative orientations of the subunits within the pentamer. This suggests that the antagonist-bound AChBP pentamer does not undergo the 15–16° rigid-body rotation of the inner region of subunits proposed as a mechanism for agonist activation of the nAChR (Unwin et al, 2002). Detailed view of the Cbtx–AChBP complex interface Three distinct, separated anchor points of Cbtx on AChBP result in a very well-ordered bound toxin molecule (Figures 4 and 6), consistent with its nanomolar dissociation constant for AChBP (Hansen et al, 2002). The three regions of Cbtx responsible for complex formation, loops I and II and part of the C-terminus, are located in the concave face of the molecule with contributions from one residue and the fifth disulfide bridge located in the convex face of loop II (Table II). Cbtx loop II, which is made of residues Tyr21–Gly40 and forms a narrow hairpin with a bulbous tip containing the fifth disulfide bridge, is central to the complex interface. The Phe29 and Arg33 side chains extending at its tip are well positioned for establishing hydrophobic and aromatic interactions with AChBP Trp53, Tyr185, Tyr192, and perhaps Trp143, in the ligand-binding pocket, 10 Å into the interfacial cleft. In addition, Cbtx Trp25, Asp27, Ala28, and Ile32 that surround the tip of loop II are within contact distance of Tyr185 in AChBP subunit A and Glu163, Glu55, Leu112, Met114, and Tyr164 in subunit B. Significant side-chain interactions may also occur between Cbtx Ser31, Cys26, and Cys30, in the convex face of loop II, and the AChBP segment Ser159–Tyr164 in subunit B; yet part of this region remains disordered as found in previous AChBP structures (Celie et al, 2004). Remarkably, the position of Cbtx Phe29 in the complex overlays with that of Tyr185 in the closed loop C of Hepes-bound AChBP, as to conform to a filled binding pocket (Figures 4C and 5B). In turn, the newly positioned Tyr185 in the open loop C is sandwiched between the Cbtx Phe29 and Arg36 side chains, where it is most likely hydrogen-bonded to Cbtx Asp27 and in cation-π interaction with Arg36, instead of Arg33 as predicted from docking (Zeng and Hawrot, 2002). Finally, the Cbtx Phe29 and Arg33 side chains, respectively positioned on the principal and complementary faces of the interface, are oriented toward the AChBP binding pocket where they would partially overlap with a bound nicotine molecule; this overlap is consistent with competitive antagonism by α-neurotoxins. Yet in the Cbtx complex, the Arg33 guanidinium nearly overlays with the carbamoyl/acetyl moiety of carbamoylcholine/acetylcholine, while in the agonist-bound structures, the quaternary ammonium of the ligand is in cation-π and electrostatic interactions with the Trp143 indole and backbone carbonyl group, respectively (Celie et al, 2004). The precise orientation of Cbtx loop II at the binding interface governs the other two interaction points between Cbtx and AChBP (Figures 3 and 6; Table II). At the tip of Cbtx loop I, residues Ile5–Asp8 abut apical to the AChBP newly positioned loop C as to establish interactions with Thr184, Ala191, and Glu190 located within the β9–β10 loop. In the C-terminal region of Cbtx, the Phe65 side chain establishes interactions with AChBP loop C residues. Yet the toxin C-terminus, which contains three vicinal positively charged side chains after the bent hinge of Pro66 (Figure 1B), is disordered and appears nonessential for binding. Similarly, residues at the tip of Cbtx loop III do not contribute interactions, with the closest distance, 8 Å, between the Cα positions of Cbtx Gly51 and AChBP Asp160. This is consistent with structure–activity studies showing that the C-terminal 67–71 stretch and residues within loop III weakly contribute to Cbtx binding (Fruchart-Gaillard et al, 2002). Overall, this three-point mode of binding closely resembles that previously observed for Fas2 bound to AChE (Bourne et al, 1995). However, while Fas2 sterically occludes substrate entry to the AChE active site gorge, Cbtx prevents binding of small ligands by binding to an overlapping region in the AChBP binding pocket and promoting significant conformational changes. Conformational changes in AChBP upon toxin binding AChBP loops C and F are major components of the principal and complementary faces of the subunit interface. In the complex, the loop C tip, comprised of residues Tyr185–Tyr192 and the Cys187–Cys188 disulfide bridge (Figure 1A), is markedly dislodged from its close apposition to the complementary face of the subunit interface seen in Hepes-bound AChBP (Brejc et al, 2001) (Figure 5). The 10 Å distance between the two positions results from a 35° rotation of the loop around an axis that roughly aligns with a vector connecting Ser182 to Glu193. The newly positioned loop C is located midway between Cbtx loops I and II where it is sequestered and almost entirely wrapped by the Cbtx concave face and C-terminal region (Figure 6A). Compared to the closed conformation of loop C, the backbone trace significantly deviates from Ser182 to Tyr192 where two major hinge positions for structural rearrangement are found within the Ser182–Val183 and Ala191–Tyr192 dipeptides. An additional change in conformation for the Cbtx-bound relative to the Hepes-bound AChBP occurs in loop F where the solvent-exposed Thr155–Tyr164 segment, which faces loop C at the cleft entrance, moves toward strand β9 with the largest deviation, up to 3.5 Å, found for Glu163. Although the Thr155–Ser159 backbone trace is not resolved in d