NMR structure determination of a novel conotoxin, [Pro 7,13] αA-conotoxin Piva

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NMR structure determination of a novel conotoxin, [Pro 7,13] αA-conotoxin Piva
Kyou Hoon Han; Kae-Jung Hwang; Seung Moak Kim; Soo-Kyung Kim; W R Gray; B M Olivera; J Rivier; Ki-Joon Shon
Bibliographic Citation
Biochemistry, vol. 36, no. 7, pp. 1669-1677
Publication Year
A high-resolution solution conformation of a novel conotoxin, [Pro 7,13] αA-conotoxin P(IVA), GCCGSYPNAACHPCSCKDROSYCGQ-NH2, has been determined by two-dimensional 1H NMR methods and distance geometry calculations. The total of 324 NOE-derived interproton distance restraints including 33 long-range NOE restraints as well as 11 φ and 7 χ1 torsion angle restraints was used for computation of structures. Back-calculation from the experimental NOE spectrum has provided 49 new NOE restraints and yielded the final R-factors of R(a) = 0.641 and R(b), = 0.157. The final RMSD values are 0.90 and 1.16 ? for the backbone and the heavy atoms, respectively. The C-terminal half of the molecule involving the residues 12-24 is extremely well-defined with a backbone RMSD value of 0.56 ?, whereas the N-terminal 3-11 disulfide loop is relatively flexible, possessing a backbone RMSD value of 1.09 ?. The [Pro 7,13] αA-conotoxin P(IVA) does not contain any significant secondary structure although the 21S-24G nearly completes one turn of a 310 helix. The overall protein fold is largely maintained by the three disulfide bridges of 2-16, 3-11, and 14-23. The presence of the three disulfide bridges imposes geometric constraints that force the molecule to form six continuous bends involving the following residues: 3C-5S, 7P-10A, 12H-14C, 15S-17K, 17K-19R, and 21S-25Q. The overall shape of the [Pro 7,13] αA-conotoxin P(IVA) can be described as an 'iron'. Residues 15S-19R form a loop that protrudes out of the 'bottom plate' formed by the rest of the protein and constitute the handle of the iron. The N-terminal tip of the molecule is relatively immobile due to attractive electrostatic interactions between the γ-hydroxyl group of 20 Hyp and the phenolic hydroxyl group of 22Y. The flexible 3-11 disulfide loop consists mostly of hydrophobic residues, while the best defined 14-23 disulfide loop contains the highly charged hydrophilic 15S-19R 'handle' domain exposed to the exterior of the protein. Binding to nicotinic acetylcholine receptor can be mediated through two different types of interactions: one involving the aromatic hydrophobic residues such as 6Y and 12H and the other involving the positively charged hydrophilic side chain of the 19R. The side chain of the 19R in the [Pro 7,13] αA-conotoxin P(IVA) and that of the 9R of the α-conotoxin G1, and also the side chains of the 12H and 6Y in the former and those of 10H and 11Y in the latter can be aligned to point to the same direction when the corresponding backbone atoms are superimposed to an RMSD value of 2.5 ?.
Amer Chem Soc
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