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• W2026280346 abstract "We report the solution three-dimensional structure of an αA-conotoxin EIVA determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The αA-conotoxin EIVA consists of 30 amino acids representing the largest peptide among the α/αA-family conotoxins discovered so far and targets the neuromuscular nicotinic acetylcholine receptor with high affinity. αA-Conotoxin EIVA consists of three distinct structural domains. The first domain is mainly composed of the Cys3-Cys11-disulfide loop and is structurally ill-defined with a large backbone root mean square deviation of 1.91 Å. The second domain formed by residues His12-Hyp21 is extremely well defined with a backbone root mean square deviation of 0.52 Å, thus forming a sturdy stem for the entire molecule. The third C-terminal domain formed by residues Hyp22-Gly29 shows an intermediate structural order having a backbone root mean square deviation of 1.04 Å. A structurally ill-defined N-terminal first loop domain connected to a rigid central molecular stem seems to be the general structural feature of the αA-conotoxin subfamily. A detailed structural comparison between αA-conotoxin EIVA and αA-conotoxin PIVA suggests that the higher receptor affinity of αA-conotoxin EIVA than αA-conotoxin PIVA might originate from different steric disposition and charge distribution in the second loop “handle” motif. We report the solution three-dimensional structure of an αA-conotoxin EIVA determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The αA-conotoxin EIVA consists of 30 amino acids representing the largest peptide among the α/αA-family conotoxins discovered so far and targets the neuromuscular nicotinic acetylcholine receptor with high affinity. αA-Conotoxin EIVA consists of three distinct structural domains. The first domain is mainly composed of the Cys3-Cys11-disulfide loop and is structurally ill-defined with a large backbone root mean square deviation of 1.91 Å. The second domain formed by residues His12-Hyp21 is extremely well defined with a backbone root mean square deviation of 0.52 Å, thus forming a sturdy stem for the entire molecule. The third C-terminal domain formed by residues Hyp22-Gly29 shows an intermediate structural order having a backbone root mean square deviation of 1.04 Å. A structurally ill-defined N-terminal first loop domain connected to a rigid central molecular stem seems to be the general structural feature of the αA-conotoxin subfamily. A detailed structural comparison between αA-conotoxin EIVA and αA-conotoxin PIVA suggests that the higher receptor affinity of αA-conotoxin EIVA than αA-conotoxin PIVA might originate from different steric disposition and charge distribution in the second loop “handle” motif. The fish-hunting cone snail Conus ermineus is the only known piscivorous Conus species in the Atlantic Ocean (1Walls J.G. Cone Shells. A Synopsis of the Living Conidae. T. F. H. Publications Inc. Ltd., Hong Kong1979Google Scholar, 2Abbott R.R. Dance S.P. Compendium of Seashells. American Malacologists Inc., Melbourne, Florida1986Google Scholar). In aquaria, the species captures its prey by extending its highly distensible amber-colored proboscis from which ejected is a hollow harpoon-shaped tooth that functions as a hypodermic needle to inject venom into the fish. Venom injection results in a complete inhibition of neuromuscular transmission in the injected prey. A major molecular component of this physiological strategy is to prevent neurotransmitter (acetylcholine) binding to the major postsynaptic receptor, the muscle subtype of the nicotinic acetylcholine receptor. In most fish-hunting cone snail venoms, the competitive nicotinic antagonists of the skeletal muscle subtype are peptides with two disulfide bonds belonging to the α-conotoxin family (3McIntosh J.M. Santos A.D. Olivera B.M. Annu. Rev. Biochem. 1999; 68: 59-88Crossref PubMed Scopus (278) Google Scholar). However, in C. ermineus, there are two different nicotinic receptor antagonists: 1) α-conotoxin EI with two disulfide bonds (4Martinez J.S. Olivera B.M. Gray W.R. Craig A.G. Groebe D.R. Abramson S.N. McIntosh J.M. Biochemistry. 1995; 34: 14519-14526Crossref PubMed Scopus (104) Google Scholar) and 2) αA-conotoxin EIVA with three disulfide bonds (5Jacobsen R. Yoshikami D. Ellison M. Martinez J. Gray W.R. Cartier G.E. Shon K.-J. Groebe D.R. Abramson S.N. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1997; 272: 22531-22537Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The αA-conotoxins have been found in only two Conus species: 1) C. ermineus from the Atlantic and 2) the eastern Pacific purple cone, Conus purprascens (6Hopkins C. Grilley M. Miller C. Shon K.-J. Cruz L.J. Gray W.R. Dyker J. Rivier J. Yoshikami D. Olivera B.M. J. Biol. Chem. 1995; 270: 22361-22367Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). These species are believed to have a different evolutionary history from the Indo-Pacific fish-hunting Conus (7Espiritu D.J.D. Watkins M. Dia-Monje V. Cartier G.E. Cruz L.J. Olivera B.M. Toxicon. 2001; 39: 1899-1916Crossref PubMed Scopus (146) Google Scholar, 8Röckel D. Korn W. Kohn A.J. Manual of the Living Conidae, Vol. I. Indo-Pacific Region, Verlag Christa Hemmen, Wiesbaden, Germany1995Google Scholar). Highly selective antagonistic activity against particular subtypes of nicotinic acetylcholine receptors (nAChRs) 1The abbreviations used are: nAChR, nicotinic acetylcholine receptor; r.m.s., root mean square; COSY, shift correlation spectroscopy; TOCSY, total COSY; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy. (Table I) has rendered α/αA-conotoxins an important tool for studying molecular function of nAChR (3McIntosh J.M. Santos A.D. Olivera B.M. Annu. Rev. Biochem. 1999; 68: 59-88Crossref PubMed Scopus (278) Google Scholar, 9Myers R.A. Cruz L.J. Rivier J.E. Olivera B.A. Chem. Rev. 1993; 93: 1923-1936Crossref Scopus (186) Google Scholar, 10Dutton J.L. Craik D.J. Curr. Med. Chem. 2001; 8: 327-344Crossref PubMed Scopus (97) Google Scholar, 11Arias H.R. Blanton M.P. Int. J. Biochem. Cell Biol. 2000; 32: 1017-1028Crossref PubMed Scopus (67) Google Scholar). In an effort to discover novel modulators of nAChR by identifying critical residues within the toxin for receptor contact, we have been applying a “reverse mapping strategy” using atomic resolution structures of various α/αA-conotoxins (12Han K.-H. Hwang K.-J. Kim S.-M. Kim S.-K. Gray W.R. Olivera B.M. Rivier J. Shon K.-J. Biochemistry. 1997; 36: 1669-1677Crossref PubMed Scopus (32) Google Scholar, 13Mok K.H. Han K.-H. Biochemistry. 1999; 38: 11895-11904Crossref PubMed Scopus (17) Google Scholar, 14Cho J. Mok K.H. Olivera B.M. McIntosh J.M. Park K.-H. Han K.-H. J. Biol. Chem. 2000; 275: 8680-8685Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15Park K.-H. Suk J.-E. Jacobsen R. Gray W.R. McIntosh J.M. Han K.-H. J. Biol. Chem. 2001; 276: 49028-49033Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Although considerable structure-function work has been reported on the α-conotoxins (16Guddat L.W. Martin J.A. Shan L. Edmunson A.B. Gray W.R. Biochemistry. 1996; 35: 11329-11335Crossref PubMed Scopus (91) Google Scholar, 17Hu S.-H. Gehrmann J. Guddat L.W. Alewood P.F. Craik D.J. Martin J.L. Structure. 1996; 4: 417-423Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 18Hu S.-H. Gehrmann J. Alewood P.F. Craik D.J. Martin J.L. Biochemistry. 1997; 36: 11323-11330Crossref PubMed Scopus (80) Google Scholar, 19Shon K.-J. Koerber S.C. Rivier J.E. Olivera B.M. McIntosh J.M. Biochemistry. 1997; 36: 15693-15700Crossref PubMed Scopus (57) Google Scholar, 20Hu S.-H. Loughnan M. Miller R. Weeks C.M. Blessing R.H. Alewood P.F. Lewis R.J. Martin J.L. Biochemistry. 1998; 37: 11425-11433Crossref PubMed Scopus (53) Google Scholar, 21Maslennikov I.V. Shenkarev Z.O. Zhmak M.N. Ivanov V.T. Methfessel C. Tsetlin V.I. Arseniv A.S. FEBS Lett. 1999; 444: 275-280Crossref PubMed Scopus (63) Google Scholar, 22Favreau P. Krimm I. Le Gall F. Bobenrieth M.-J. Lamthanh H. Bouet F. Servent D. Molgo J. Menez A. Letourneux Y. Lancelin J.-M. Biochemistry. 1999; 38: 6317-6326Crossref PubMed Scopus (59) Google Scholar, 23Lamthanh H. Jegou-Matheron C. Servent D. Menez A. Lancelin J.M. FEBS Lett. 1999; 454: 293-298Crossref PubMed Scopus (47) Google Scholar, 24Rogers J.P. Luginbuhl P. Shen G.S. McCabe R.T. Stevens R.C. Wemmer D.E. Biochemistry. 1999; 38: 3874-3882Crossref PubMed Scopus (49) Google Scholar, 25Benie A.J. Whitford D. Hargittai B. Barany G. Janes R.W. FEBS Lett. 2000; 476: 287-295Crossref PubMed Scopus (23) Google Scholar), there has been only one report on the αA-subfamily (12Han K.-H. Hwang K.-J. Kim S.-M. Kim S.-K. Gray W.R. Olivera B.M. Rivier J. Shon K.-J. Biochemistry. 1997; 36: 1669-1677Crossref PubMed Scopus (32) Google Scholar) since their original discovery (6Hopkins C. Grilley M. Miller C. Shon K.-J. Cruz L.J. Gray W.R. Dyker J. Rivier J. Yoshikami D. Olivera B.M. J. Biol. Chem. 1995; 270: 22361-22367Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). αA-Conotoxin EIVA is different in its pharmacological specificity from the best studied α-conotoxins (such as α-conotoxin MI) in that it will inhibit both ligand-binding sites on the muscle nicotinic receptor with an approximately equal affinity (5Jacobsen R. Yoshikami D. Ellison M. Martinez J. Gray W.R. Cartier G.E. Shon K.-J. Groebe D.R. Abramson S.N. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1997; 272: 22531-22537Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) instead of being highly selective either for the α1/δ or for the α1/γ interface of heteropentameric neuromuscular nAChRs. In this article, we report the high resolution NMR structure of αA-conotoxin EIVA from C. ermineus that exhibits the highest (subnanomolar) receptor affinity among all of the known nAChR-targeting conotoxins.Table IThree subfamilies of nAChR-targeting conotoxins Peptide Preparation—αA-Conotoxin EIVA, originally purified from the venom of C. ermineus, was synthesized and purified as described previously (5Jacobsen R. Yoshikami D. Ellison M. Martinez J. Gray W.R. Cartier G.E. Shon K.-J. Groebe D.R. Abramson S.N. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1997; 272: 22531-22537Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Disulfide-bonding patterns were assigned by mass spectrometry as well as by NMR. When αA-conotoxin EIVA was analyzed by liquid-secondary ionization mass spectrometry, a monoisotopic mass of 3095.2 was obtained (5Jacobsen R. Yoshikami D. Ellison M. Martinez J. Gray W.R. Cartier G.E. Shon K.-J. Groebe D.R. Abramson S.N. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1997; 272: 22531-22537Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), suggesting the presence of three disulfide bonds. In addition, the formation of three specific disulfide bonds in αA-conotoxin EIVA was unambiguously determined by using a well established NMR protocol that relies on the fact that NOEs involving relevant α and β protons of pairing cysteines are observed for a disulfide bond (26Klaus W. Broger C. Gerber P. Senn H. J. Mol. Biol. 1993; 232: 897-906Crossref PubMed Scopus (103) Google Scholar) (for details see “Structure Calculations”). NMR Experiments—Samples for the NMR studies were prepared in 90% H2O, 10% 2H2O or in 100% 2H2O with a final concentration of ∼5 mm at pH 3.6. The pH was measured as a direct reading from a combination microelectrode calibrated at two reference pH values. All of the NMR experiments were performed using a Varian UNITY 500 or UNITY INOVA 600 spectrometer at three temperatures to obtain unambiguous resonance assignment. For TOCSY experiments (27Griesinger C. Otting G. Wüthrich K. Ernst R.R. J. Am. Chem. Soc. 1988; 110: 7870-7872Crossref Scopus (1199) Google Scholar), mixing times of 65–87 ms were applied. All of the peaks were referenced to a residual water signal (4.76 ppm at 25 °C). Spectral widths were 6 kHz in both dimensions. Typical two-dimensional data consist of 2048 complex points in the t 2 dimension with 256 complex t 1 increments. Structure Calculations—Interproton distance restraints used for computation of structures were derived primarily from the NOESY spectrum recorded with a mixing time of 200 ms obtained at 15 °C. Several long range Hα-Hβ NOEs (residues 3–11, 11–3, 14–24, 16–2, and 24–14) and Hβ-Hβ NOEs (residues 2–16, 3–11, and 14–24) were clearly observed, confirming the presence of three disulfide bonds, Cys2-Cys16, Cys3-Cys11, and Cys14-Cys24. The FELIX program in the NMR Refine module of Biosym 95.0 software (Molecular Simulations Inc., San Diego, CA) was used for quantification of NOE volumes and for converting them into interproton distance restraints. As a distance reference, the NOE volumes of four non-overlapping geminal β-proton cross-peaks were averaged and correlated with the appropriate geminal distance of 1.8 Å. Volume integration errors and influence of possible conformational averaging were taken into consideration by adding 0.5 and 1.0 Å to distance restraints involving only backbone protons and to those containing at least one side chain proton, respectively (28Wüthrich K. Billeter M. Braun W. J. Mol. Biol. 1983; 169: 949-961Crossref PubMed Scopus (1007) Google Scholar). Actual computation of structures was done in two major steps. The first involved the generation of 50 low resolution structures using DGII calculation based on the metric-matrix method (Molecular Simulations Inc.). For example, to refine the initial distance restraints and to obtain more accurate distance restraints by correcting for spin-diffusion effect, the RANDMARDI program (29Liu H. Sielmann H.P. Ulyanov N.B. Wemmer D.E. James T.L. J. Biomol. NMR. 1995; 6: 390-402Crossref PubMed Scopus (110) Google Scholar) was run for the structures generated by DGII. The final restraint set included a total of 388 restraints, 357 of which were NOE-derived distance restraints that in turn were composed of 164 intraresidue distances, 152 short range (|i–j| < 5) interresidue distances, and 41 long range (|i–j| > 5) interresidue distances. Also included in the restraint file were 11 torsion angle restraints along with 11 chirality and 9 disulfide-bond constraints. Backbone torsion angle ϕ was set to -120° (±30°) when 3 J HNHα > 8 Hz, and backbone torsion angle ϕ is set to -60° (±30°) if 3 J HNHα < 6 Hz. The second step of structure calculation was refinement of the DGII-generated structures by restrained molecular dynamics (30Clore G.M. Sukumaran D.K. Nilges M. Gronenborn A.M. Biochemistry. 1987; 26: 1732-1745Crossref Scopus (65) Google Scholar) done in a biphasic manner such that distance restraints associated with the backbone conformation were applied during the first 22 ps of dynamics at 1000 K followed by application of the rest of distance restraints. Each dynamics run lasted for 84 ps, 52 ps at 1000 K and 32 ps of annealing period down to 100 K, finally followed by a short energy minimization using a conjugate gradient method. Force constants for the NOE restraints and for torsion angles were gradually increased to the final values of 30 kcal/mol. NMR Spectroscopy—Complete 1H resonance assignment for the αA-conotoxin EIVA was achieved using homonuclear two-dimensional NMR methods according to the standard sequential resonance assignment strategy. Typically, classification of spin systems in a TOCSY spectrum was followed by the sequential resonance assignment procedure based upon sequential NOE connectivity (31Billeter M. Braun W. Wüthrich K. J. Mol. Biol. 1982; 155: 321-346Crossref PubMed Scopus (567) Google Scholar). Fig. 1 shows the finger-print region in the NOESY spectrum of αA-conotoxin EIVA obtained in 90% H2O, 10% 2H2O at pH 3.6, and summarized in Fig. 2 are short- and medium-range NOEs, 3 J HNHα, and chemical shift index (32Wishart D.S. Sykes B.D. Methods Enzymol. 1994; 239: 363-392Crossref PubMed Scopus (939) Google Scholar) along the amino acid sequence of the αA-conotoxin EIVA. For some residues such as His12, Lys17, Arg20, and Arg26, the presence of unique “back-transfer” cross-peaks in the TOCSY spectrum (data not shown) helped to obtain unambiguous resonance assignment readily without relying on the NOE connectivity information.Fig. 2Amino acid sequence of αA-conotoxin EIVA and a summary of short- and medium-range NOEs, 3 J HNHα, and chemical shift index (CSI) for Hα protons. Thickness of bar represents relative strength (strong, medium, and weak) of NOEs. Filled circles are drawn when 3 J HNHα < 6 Hz, and open circles are drawn when 3 J HNHα > 8 Hz. The filled squares above and below the horizontal line represent CSI values of +1 and -1, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Structural Description of αA-Conotoxin EIVA— Fig. 3 shows the ensemble of 18 final converged structures of αA-conotoxin EIVA. The global molecular fold is stabilized mainly by three disulfide bridges of Cys2-Cys16, Cys3-Cys11, and Cys14-Cys24 without any distinct secondary structural elements. From a structural standpoint, αA-conotoxin EIVA consists of three distinct structural domains. The first domain is mainly composed of residues in the Cys3-Cys11 loop and exhibits a high conformational disorder with a backbone r.m.s. deviation of 1.91 Å. The Cα T 1 and T 2 relaxation times for some residues (Cys2, Pro5, Ala9, Ala10, and Cys11) in the first domain are on the order of ∼350 and ∼100 ms, respectively, with the exception of the Pro5. These numbers do not differ significantly from the Cα T 1 and T 2 values of the residues in the other domains, indicating that the conformational disorder observed for the first domain is most probably because of the absence of appropriate NOE restraints, in particular, near the Gly4-Pro5-Tyr6-Hyp7 segment. This first domain is connected to an extremely well defined loop domain formed by residues His12-Hyp21 that has a backbone r.m.s. deviation of 0.52 Å and corresponds to the “handle” of an iron (see below). The third C-terminal domain consists of residues Hyp22-Gly29 and shows an intermediate structural order with a backbone r.m.s. deviation of 1.04 Å. The presence of such three distinct structural domains appears to be the common feature of αA-conotoxins (12Han K.-H. Hwang K.-J. Kim S.-M. Kim S.-K. Gray W.R. Olivera B.M. Rivier J. Shon K.-J. Biochemistry. 1997; 36: 1669-1677Crossref PubMed Scopus (32) Google Scholar). In Fig. 4, GRASP images of αA-conotoxin EIVA (left) and αA-conotoxin PIVA (right) are shown. The overall shape of αA-conotoxin PIVA was previously shown to resemble an “iron” with the Gln25 located at the forwarding tip of an iron (12Han K.-H. Hwang K.-J. Kim S.-M. Kim S.-K. Gray W.R. Olivera B.M. Rivier J. Shon K.-J. Biochemistry. 1997; 36: 1669-1677Crossref PubMed Scopus (32) Google Scholar). In the case of αA-conotoxin EIVA, however, Arg26 assumes the corresponding position. The C-terminal additional four residues, Hyp27-Ser28-Gly29-Gly30, of αA-conotoxin EIVA seem to attach themselves as a mobile anchor to the molecular body (Fig. 4, left top). In Fig. 4, the three residues (Lys17-Asp18-Arg19) in αA-conotoxin PIVA protrude toward the reader out of the “bottom plate” of an iron that is formed by the rest of the molecule. In contrast, four residues of αA-conotoxin EIVA, Lys17-Val18-Gly19-Arg20, form a rigid loop in the central stem of αA-conotoxin EIVA. Detailed structural comparison of these loops is shown in Fig. 5 where the protruding loop in αA-conotoxin EIVA shows more hydrophobic character than the corresponding loop in αA-conotoxin PIVA.Fig. 5Superposition of the protruding loops in two αA-conotoxins. One (purple) is composed of Lys17-Val18-Gly19-Arg20 (αA-conotoxin EIVA) and the other (cyan) is composed of Lys17-Asp18-Arg19 (αA-conotoxin PIVA). Both loops are located in the “handle” portion of an iron. For visual clarity, only selected residues are labeled. Note the excellent backbone superposition between two molecules up to the residue Lys17 and then deviation from the 18th residue.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Table II is a summary of structure determination statistics. The average r.m.s. deviation values for backbone (0.66 Å) and heavy atoms (1.33 Å) within the ensemble represent typical values for reasonably well determined structures. Reasonable quality of structures can also be inferred from the two R-factors (33Thomas P.D. Basus V.J. James T.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1237-1241Crossref PubMed Scopus (184) Google Scholar, 34Borgias B.A. James T.L. J. Magn. Res. 1988; 79: 493-512Google Scholar), R a = 0.607 and R b = 0.204, as well as the results of PROCHECK analysis (35Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Note that the angular order parameters for the backbone torsion angles become very high when the structurally ill-defined N-terminal domain of the molecule is excluded.Table IINMR structure determination statistics of αA-conotoxin EIVA for an ensemble of 18 structuresR.m.s. deviations from experimental restraintsaValues where applicable are the means ± S.DInterproton distances (Å)0.0657 ± 0.0059Torsion angle violation (°)0.3013 ± 0.0823Ramachandran plot statisticsResidues in most favored region (%)61.1Residues in additionally allowed region (%)33.3Residues in generously allowed region (%)5.6Residues in disallowed region (%)0.0Ensemble R factorsbRa and Rb values are computed as defined in Ref. 33Ra0.607 ± 0.013Rb0.204 ± 0.006Angular order parametersφ0.9096 ± 0.0957ψ0.8080 ± 0.2326χ10.6785 ± 0.3299φ (residue 11—25)0.9474 ± 0.0357ψ (residue 11—25)0.9681 ± 0.0349χ1 (residue 11—25)0.8033 ± 0.2631R.m.s. deviations from the average structure (residues 11—25)Backbone atomscBackbone atoms are N, Cα, C′, and O (Å)0.66Heavy atoms (Å)1.33a Values where applicable are the means ± S.Db Ra and Rb values are computed as defined in Ref. 33Thomas P.D. Basus V.J. James T.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1237-1241Crossref PubMed Scopus (184) Google Scholarc Backbone atoms are N, Cα, C′, and O Open table in a new tab Two nAChR antagonist peptides produced by the Atlantic cone shell C. ermineus, α-conotoxin EI (4Martinez J.S. Olivera B.M. Gray W.R. Craig A.G. Groebe D.R. Abramson S.N. McIntosh J.M. Biochemistry. 1995; 34: 14519-14526Crossref PubMed Scopus (104) Google Scholar, 15Park K.-H. Suk J.-E. Jacobsen R. Gray W.R. McIntosh J.M. Han K.-H. J. Biol. Chem. 2001; 276: 49028-49033Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and αA-conotoxin EIVA (5Jacobsen R. Yoshikami D. Ellison M. Martinez J. Gray W.R. Cartier G.E. Shon K.-J. Groebe D.R. Abramson S.N. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1997; 272: 22531-22537Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), are both peculiar. The former selectively antagonizes neuromuscular receptor despite the fact that it belongs to the α4/7 2The α-conotoxins are grouped according to the number of amino acid residues enclosed within each disulfide loop. Loop sizes of three residues in the first loop and five in the second are denoted as α3/5. Other subfamilies include α4/7 and α4/3. subfamily, all of the other members of which target neuronal subtypes (Table I). In addition, it is the only nAChR-antagonizing conotoxin that shows a high affinity toward the α1/δ site of the Torpedo nAChR. All of the other neuromuscular toxins either show a high affinity for the α1/γ site or do not distinguish between the two sites. The second nAChR antagonist αA-conotoxin EIVA produced by C. ermineus, whose three-dimensional structure is presented in the current report, is unique in other ways. αA-Conotoxin EIVA targets nAChR, not Na+ or Ca2+ channels, as do the other three disulfide-constrained conotoxins such as μ- or ω-conotoxins (3McIntosh J.M. Santos A.D. Olivera B.M. Annu. Rev. Biochem. 1999; 68: 59-88Crossref PubMed Scopus (278) Google Scholar, 4Martinez J.S. Olivera B.M. Gray W.R. Craig A.G. Groebe D.R. Abramson S.N. McIntosh J.M. Biochemistry. 1995; 34: 14519-14526Crossref PubMed Scopus (104) Google Scholar, 6Hopkins C. Grilley M. Miller C. Shon K.-J. Cruz L.J. Gray W.R. Dyker J. Rivier J. Yoshikami D. Olivera B.M. J. Biol. Chem. 1995; 270: 22361-22367Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 9Myers R.A. Cruz L.J. Rivier J.E. Olivera B.A. Chem. Rev. 1993; 93: 1923-1936Crossref Scopus (186) Google Scholar). Interestingly, αA-conotoxin EIVA is incapable of discriminating between the α1/δ site and the α1/γ site in contrast to the other neuromuscular conotoxins such as α3/5-conotoxins or α-conotoxin EI. Efforts to gain a clear understanding of molecular function of nAChR would be greatly aided by a high resolution three-dimensional structure of nAChR. Unfortunately, attempts to obtain such information have not been successful (36Grant M.A. Gentile L.N. Shi Q.-L. Pellegrini M. Hawrot E. Biochemistry. 1999; 38: 10730-10742Crossref PubMed Scopus (13) Google Scholar, 37Alexeev T. Krivoshein A. Shevalier A. Kudelina I. Telyakova O. Vincent A. Utkin Y. Hucho F. Tsetlin V. Eur. J. Biochem. 1999; 259: 310-319Crossref PubMed Scopus (33) Google Scholar, 38Yao Y. Wang J. Viroonchatapan N. Samson A. Chill J. Rothe E. Anglister J. Wang Z.-Z. J. Biol. Chem. 2002; 277: 12613-12621Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) even though a low resolution electromagnetic image (39Unwin N. J. Mol. Biol. 1996; 257: 586-596Crossref PubMed Scopus (99) Google Scholar, 40Miyazawa A. Fujiyoshi Y. Stowell M. Unwin N. J. Mol. Biol. 1999; 288: 765-786Crossref PubMed Scopus (430) Google Scholar) and an x-ray structure of an acetylcholine-binding protein that resembles the extracellular domain of nAChR have been obtained (41Brejc K. Dijk W.J. Klaaxxen R.V. Scheuurmans M. Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1586) Google Scholar). In the absence of a high resolution structure of nAChR, we have been determining high resolution structures of α/αA-conotoxins and tried to “reverse-predict” the receptor residues that might interact with the receptor-contacting residues of ligands (12Han K.-H. Hwang K.-J. Kim S.-M. Kim S.-K. Gray W.R. Olivera B.M. Rivier J. Shon K.-J. Biochemistry. 1997; 36: 1669-1677Crossref PubMed Scopus (32) Google Scholar, 13Mok K.H. Han K.-H. Biochemistry. 1999; 38: 11895-11904Crossref PubMed Scopus (17) Google Scholar, 14Cho J. Mok K.H. Olivera B.M. McIntosh J.M. Park K.-H. Han K.-H. J. Biol. Chem. 2000; 275: 8680-8685Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15Park K.-H. Suk J.-E. Jacobsen R. Gray W.R. McIntosh J.M. Han K.-H. J. Biol. Chem. 2001; 276: 49028-49033Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Such an approach is possible because although various α/αA-conotoxins exhibit a great variety of receptor subtype specificity, they differ only slightly in their sequences with a similar overall fold exhibiting subtle local structural differences. Two useful features have emerged from structural studies on α/αA-conotoxins. First, potential receptor contact residues within each toxin have been suggested (12Han K.-H. Hwang K.-J. Kim S.-M. Kim S.-K. Gray W.R. Olivera B.M. Rivier J. Shon K.-J. Biochemistry. 1997; 36: 1669-1677Crossref PubMed Scopus (32) Google Scholar, 14Cho J. Mok K.H. Olivera B.M. McIntosh J.M. Park K.-H. Han K.-H. J. Biol. Chem. 2000; 275: 8680-8685Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15Park K.-H. Suk J.-E. Jacobsen R. Gray W.R. McIntosh J.M. Han K.-H. J. Biol. Chem. 2001; 276: 49028-49033Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). High resolution structures have also helped us to visualize how receptor residues that had been identified by other means such as affinity labeling and mutagenesis (42Galzi J.L. Revah F. Black D. Goeldner M. Hirth C. Changeux J.-P. J. Biol. Chem. 1990; 265: 10430-10437Abstract Full Text PDF PubMed Google Scholar, 43Chiara D.C. Cohen J.B. J. Biol. Chem. 1997; 272: 32940-32950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 44Sugiyama N. Marchot P. Kawanishi C. Osaka H. Molles B. Sine S.M. Taylor P. Mol. Pharmacol. 1998; 53: 787-794Crossref PubMed Scopus (42) Google Scholar, 45Chiara D.C. Xie Y. Cohen J.B. Biochemistry. 1999; 38: 6689-6698Crossref PubMed Scopus (59) Google Scholar, 46Osaka H. Malany S. Molles B.E. Sine S.M. Taylor P. J. Biol. Chem. 2000; 275: 5478-5484Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 47Xie Y. Cohen J.B. J. Biol. Chem. 2001; 276: 2417-2426Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) could be spatially positioned with respect to the receptor-contacting residues of ligands in order to be properly engaged in π-cation or hydrophobic interactions that are known to be major driving forces for ligand-nAChR binding (48Ma J.C. Dougherty D.A. Chem. Rev. 1997; 97: 1303-1324Crossref PubMed Scopus (3342) Google Scholar, 49Quiram P.A. McIntosh J.M. Sine S.M. J. Biol. Chem. 2000; 275: 4889-4896Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 50Bren N. Sine S.M. J. Biol. Chem. 2000; 275: 12692-12700Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The second feature that emerged from structural studies of α/αA-conotoxins is the estimated size of the ligand binding pocket in nAChR (15Park K.-H. Suk J.-E. Jacobsen R. Gray W.R. McIntosh J.M. Han K.-H. J. Biol. Chem. 2001; 276: 49028-49033Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The suggested size of the ligand binding pocket of ∼20 Å (height) × 20 Å (width) × 15 Å (thickness) is in good agreement with what has been proposed by electromagnetic images (38Yao Y. Wang J. Viroonchatapan N. Samson A. Chill J. Rothe E. Anglister J. Wang Z.-Z. J. Biol. Chem. 2002; 277: 12613-12621Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 39Unwin N. J. Mol. Biol. 1996; 257: 586-596Crossref PubMed Scopus (99) Google Scholar) or by a recent homology-modeling study (51Samson A.O. Scherf T. Eisenstein M. Chill J.H. Anglister J. Neuron. 2002; 35: 319-322Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). αA-Conotoxins are larger than typical α-conotoxins and hence may need to bend or deform themselves to fit into the same ligand binding pocket of nAChR. The presence of a flexible N-terminal loop in αA-conotoxins may help such a conformational change upon receptor binding. αA-Conotoxin EIVA blocks the acetylcholine-evoked response of Torpedo neuromuscular nAChRs expressed in Xenopus oocytes with an IC50 of 17 nm (5Jacobsen R. Yoshikami D. Ellison M. Martinez J. Gray W.R. Cartier G.E. Shon K.-J. Groebe D.R. Abramson S.N. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1997; 272: 22531-22537Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). [Pro7,13] αA-Conotoxin PIVA inhibits the binding of 125I α-bungarotoxin to Torpedo membrane with an IC50 of ∼350 nm (6Hopkins C. Grilley M. Miller C. Shon K.-J. Cruz L.J. Gray W.R. Dyker J. Rivier J. Yoshikami D. Olivera B.M. J. Biol. Chem. 1995; 270: 22361-22367Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Although affinities of the two toxins were not measured by an exactly same method, it is clear that αA-conotoxin EIVA is a more potent antagonist than αA-conotoxin PIVA. There are two prominent sequence differences between the two toxins that might account for such affinity difference. First, αA-conotoxin EIVA has an additional four residues, Hyp27-Ser28-Gly29-Gly30, at the C terminus. The second difference is found in the protruding loop region. It is unknown whether both differences contribute to the affinity difference between the two toxins. Ligand-nAChR interactions are believed to be mediated mainly by π-cation or hydrophobic interactions (48Ma J.C. Dougherty D.A. Chem. Rev. 1997; 97: 1303-1324Crossref PubMed Scopus (3342) Google Scholar, 49Quiram P.A. McIntosh J.M. Sine S.M. J. Biol. Chem. 2000; 275: 4889-4896Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 50Bren N. Sine S.M. J. Biol. Chem. 2000; 275: 12692-12700Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Therefore, the differences in charge as well as hydrophobicity in the loop region shown in Figs. 4 and 5 are expected to contribute to the affinity difference between the two toxins. The overall molecular size of nAChR-binding ligands increases in the following order: acetylcholine, nicotine < tubocurare < α-conotoxins < αA-conotoxins < α-bungarotoxin. An interesting correlation seems to exist, albeit not fully proven yet, between the size of nAChR ligands and their ability to discriminate two unequivalent ligand-binding sites in the neuromuscular nAChR. As the size of the ligands increases, they tend to lose the ability to discriminate two unequivalent ligand-binding sites. For example, αA-conotoxins and α-bungarotoxins seem to become incapable of discriminating two ligand-binding sites when compared with smaller α-conotoxins or curares, although mutant forms of NmmI (52Ackermann E.J. Taylor P. Biochemistry. 1997; 36: 12836-12844Crossref PubMed Scopus (39) Google Scholar) and α-cobratoxin (53Stéphanie A.-D. Gaillard C. Tamiya T. Corringer P.-J. Changeux J.-P. Servent D. Menez A. J. Biol. Chem. 2000; 275: 29594-29601Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) are certainly exceptions to such a trend. In the case of α-bungarotoxin, it is known that the most critical receptor-contacting residues are located in its second finger (51Samson A.O. Scherf T. Eisenstein M. Chill J.H. Anglister J. Neuron. 2002; 35: 319-322Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 54Moise L. Piserchio A. Basus V.J. Hawrot E. J. Biol. Chem. 2002; 277: 12406-12417Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 55Scarselli M. Spiga O. Ciutti A. Bernini A. Bracci L. Lelli B. Lozzi L. Calamandrei D. Di Maro D. Klein S. Niccolai N. Biochemistry. 2002; 41: 1457-1463Crossref PubMed Scopus (23) Google Scholar), which is approximately similar in size to α-conotoxins. However, being a large ligand, this snake toxin may have other receptor-contacting residues that are outside the second finger such as C-terminal residues (56Basus V.J. Song G. Hawrot E. Biochemistry. 1993; 32: 12290-12298Crossref PubMed Scopus (95) Google Scholar, 57Rosenthal J.A. Levandoski M.M. Chang B. Potts J.F. Shi Q.L. Hawrot E. Biochemistry. 1999; 38: 7847-7855Crossref PubMed Scopus (34) Google Scholar). Obviously, ligand-nAChR interactions require more than one ligand-contacting “microsite” residue from the receptor with the possible number of such microsites increasing with the ligand size. The fact that αA-conotoxins cannot distinguish the two unequivalent ligand binding sites (α1/δ versus α1/γ) of neuromuscular receptor raises the possibility that binding of αA-conotoxins may involve a microsite of the nAChR that typical α-conotoxins might not touch. We thank Thomas L. James (University of California, San Francisco, CA) for generously providing the MARDIGRAS and CORMA programs." @default.
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• W2026280346 title "Solution Conformation of αA-conotoxin EIVA, a Potent Neuromuscular Nicotinic Acetylcholine Receptor Antagonist from Conus ermineus" @default.
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