DETERMINATION OF THE NMR SOLUTION STRUCTURE OF CARDIOTOXIN CTX-IIB FROM NAJA-MOSSAMBICA-MOSSAMBICA

The NMR structure of cardiotoxin CTX IIb from Naja mossambica mossambi ca in aqueous solution was determined from a total of 593 nuclear Over hauser enhancement distance constraints and 135 dihedral angle constra ints, which were collected using two-dimensional homonuclear H-1-NMR e xperiments. Structure calculations were performed with the program DIA NA, using the redundant dihedral angle constraints strategy for improv ed convergence, followed by restrained energy minimization with the pr ogram FANTOM and a modified version of the program AMBER. The CTX IIb structure is represented by a group of 20 conformers with an average r oot-mean-square deviation relative to the mean solution structure of 0 .072 nm for the backbone atoms, and 0.116 nm for all heavy atoms. The molecular structure of CTX IIb is characterized by a three-stranded be ta-sheet made up of residues 20-26, 32-39 and 48-54, and a two-strande d beta-sheet composed of residues 1-5 and 10-14. A cluster of four dis ulfide bonds, 3-21, 14-38, 42-53 and 54-59, form the core of the molec ule and crosslink the individual polypeptide strands. The NMR structur e is similar to the previously reported X-ray crystal structure of the cardiotoxin CTX V4(II) from the same species. Differences between the two structures were noted in the tips of the two loops formed by resi dues 6-9 and 27-31, which connect the beta-strand 1-5 with 10-14, and 20-26 with 32-39, respectively. For these loops the NMR data also indi cate significantly increased dynamic disorder in the solution structur e. These observations are discussed with respect to earlier suggestion s by others that these two loops are essential structural elements for function and specificity of a wide variety of homologous toxins.