NMR STRUCTURE DETERMINATION OF A NOVEL CONOTOXIN, [PRO-7,13] ALPHA-A-CONOTOXIN P-IVA

A high-resolution solution conformation of a novel conotoxin, [Pro 7,1 3] alpha A-conotoxin P-IVA, GCCGSYPNAACHPCSCKDROSYCGQ-NH2, has been de termined by two-dimensional H-1 NMR methods and distance geometry calc ulations. The total of 324 NOE-derived interproton distance restraints including 33 long-range NOE restraints as well as 11 phi and 7 chi(1) torsion angle restraints was used for computation of structures. Back -calculation from the experimental NOE spectrum has provided 49 new NO E 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 Angstrom for the back bone and the heavy atoms, respectively. The C-terminal half of the mol ecule involving the residues 12-24 is extremely well-defined with a ba ckbone RMSD value of 0.56 Angstrom, whereas the N-terminal 3-11 disulf ide loop is relatively flexible, possessing a backbone RMSD value of 1 .09 Angstrom. The [Pro 7,13] alpha A-conotoxin P-IVA does not contain any significant secondary structure although the 21S-24G nearly comple tes one turn of a 3(10) helix. The overall protein fold is largely mai ntained by the three disulfide bridges of 2-16, 3-11, and 14-23. The p resence of the three disulfide bridges imposes geometric constraints t hat force the molecule to form six continuous bends involving the foll owing residues: 3C-5S, 7P-10A, 12H-14C, 15S-17K, 17K-19R, and 21S-25Q, The overall shape of the [Pro 7,13] alpha A-conotoxin P-IVA can be de scribed as an ''iron''. Residues 15S-19R form a loop that protrudes ou t of the ''bottom plate'' formed by the rest of the protein and consti tute the handle of the iron, The N-terminal tip of the molecule is rel atively immobile due to attractive electrostatic interactions between the gamma-hydroxyl group of 20 Hyp and the phenolic hydroxyl group of 22Y. The flexible 3-11 disulfide loop consists mostly of hydrophobic r esidues, while the best-defined 14-23 disulfide loop contains the high ly charged hydrophilic 15S-19R ''handle'' domain exposed to the exteri or of the protein. Binding to nicotinic acetylcholine receptor can be mediated through two different types of interactions: one involving th e aromatic hydrophobic residues such as 6Y and 12H and the other invol ving the positively charged hydrophilic side chain of the 19R. The sid e chain of the 19R in the [Pro 7,13] alpha A-conotoxin P-IVA and that of the 9R of the alpha-conotoxin G(I), and also the side chains of the 12H and 6Y in the former and those of 10H and 11Y in the latter can b e aligned to point to the same direction when the corresponding backbo ne atoms are superimposed to an RMSD value of 2.5 Angstrom.