NMR Structure Determination of a Novel Conotoxin, [Pro 7,13] A-Conotoxin PIVA
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- 作者：Kyou-Hoon Han ; Kae-Jung Hwang ; Seung-Moak Kim ; Soo-Kyung Kim ; William R. Gray ; Baldomero M. Olivera ; Jean Rivier ; and Ki-Joon Shon
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：February 18, 1997
- 年：1997
- 卷：36
- 期：7
- 页码：1669 - 1677
- 全文大小：233K
- 年卷期：v.36,no.7(February 18, 1997)
- ISSN：1520-4995
文摘
A high-resolution solution conformation of a novel conotoxin,[Pro 7,13] 
A-conotoxin PIVA,GCCGSYPNAACHPCSCKDROSYCGQ-NH2, has been determined bytwo-dimensional 1H NMR methodsand distance geometry calculations. The total of 324 NOE-derivedinterproton distance restraints including33 long-range NOE restraints as well as 11 
and 7 
1torsion angle restraints was used for computationof structures. Back-calculation from the experimental NOE spectrumhas provided 49 new NOE restraintsand yielded the final R-factors of Ra= 0.641 and Rb = 0.157. The final RMSDvalues are 0.90 and 1.16Å for the backbone and the heavy atoms, respectively. TheC-terminal half of the molecule involving theresidues 12-24 is extremely well-defined with a backbone RMSD valueof 0.56 Å, whereas the N-terminal3-11 disulfide loop is relatively flexible, possessing a backboneRMSD value of 1.09 Å. The [Pro 7,13]A-conotoxin PIVA does not contain any significantsecondary structure although the 21S-24G nearlycompletes one turn of a 310 helix. The overall proteinfold is largely maintained by the three disulfidebridges of 2-16, 3-11, and 14-23. The presence of the threedisulfide bridges imposes geometricconstraints that force the molecule to form six continuous bendsinvolving the following residues: 3C-5S, 7P-10A, 12H-14C, 15S-17K, 17K-19R, and 21S-25Q. The overallshape of the [Pro 7,13] A-conotoxin PIVA can be described as an "iron".Residues 15S-19R form a loop that protrudes out ofthe"bottom plate" formed by the rest of the protein and constitute thehandle of the iron. The N-terminal tipof the molecule is relatively immobile due to attractive electrostaticinteractions between the 
-hydroxylgroup of 20 Hyp and the phenolic hydroxyl group of 22Y. Theflexible 3-11 disulfide loop consistsmostly of hydrophobic residues, while the best-defined 14-23disulfide loop contains the highly chargedhydrophilic 15S-19R "handle" domain exposed to the exterior of theprotein. Binding to nicotinicacetylcholine receptor can be mediated through two different types ofinteractions: one involving thearomatic hydrophobic residues such as 6Y and 12H and the otherinvolving the positively chargedhydrophilic side chain of the 19R. The side chain of the 19R inthe [Pro 7,13] A-conotoxin PIVA andthat of the 9R of the -conotoxin GI, and also the sidechains of the 12H and 6Y in the former and thoseof 10H and 11Y in the latter can be aligned to point to the samedirection when the corresponding backboneatoms are superimposed to an RMSD value of 2.5 Å.
A-conotoxin PIVA,GCCGSYPNAACHPCSCKDROSYCGQ-NH2, has been determined bytwo-dimensional 1H NMR methodsand distance geometry calculations. The total of 324 NOE-derivedinterproton distance restraints including33 long-range NOE restraints as well as 11 and 7 1torsion angle restraints was used for computationof structures. Back-calculation from the experimental NOE spectrumhas provided 49 new NOE restraintsand yielded the final R-factors of Ra= 0.641 and Rb = 0.157. The final RMSDvalues are 0.90 and 1.16Å for the backbone and the heavy atoms, respectively. TheC-terminal half of the molecule involving theresidues 12-24 is extremely well-defined with a backbone RMSD valueof 0.56 Å, whereas the N-terminal3-11 disulfide loop is relatively flexible, possessing a backboneRMSD value of 1.09 Å. The [Pro 7,13]A-conotoxin PIVA does not contain any significantsecondary structure although the 21S-24G nearlycompletes one turn of a 310 helix. The overall proteinfold is largely maintained by the three disulfidebridges of 2-16, 3-11, and 14-23. The presence of the threedisulfide bridges imposes geometricconstraints that force the molecule to form six continuous bendsinvolving the following residues: 3C-5S, 7P-10A, 12H-14C, 15S-17K, 17K-19R, and 21S-25Q. The overallshape of the [Pro 7,13] A-conotoxin PIVA can be described as an "iron".Residues 15S-19R form a loop that protrudes out ofthe"bottom plate" formed by the rest of the protein and constitute thehandle of the iron. The N-terminal tipof the molecule is relatively immobile due to attractive electrostaticinteractions between the -hydroxylgroup of 20 Hyp and the phenolic hydroxyl group of 22Y. Theflexible 3-11 disulfide loop consistsmostly of hydrophobic residues, while the best-defined 14-23disulfide loop contains the highly chargedhydrophilic 15S-19R "handle" domain exposed to the exterior of theprotein. Binding to nicotinicacetylcholine receptor can be mediated through two different types ofinteractions: one involving thearomatic hydrophobic residues such as 6Y and 12H and the otherinvolving the positively chargedhydrophilic side chain of the 19R. The side chain of the 19R inthe [Pro 7,13] A-conotoxin PIVA andthat of the 9R of the -conotoxin GI, and also the sidechains of the 12H and 6Y in the former and thoseof 10H and 11Y in the latter can be aligned to point to the samedirection when the corresponding backboneatoms are superimposed to an RMSD value of 2.5 Å.