3-DIMENSIONAL STRUCTURE IN SOLUTION OF THE POLYPEPTIDE CARDIAC STIMULANT ANTHOPLEURIN-A

The three-dimensional structure in aqueous solution of the 49-residue polypeptide anthopleurin-A (AP-A), from the sea anemone Anthopleura xa nthogramica, has been determined from H-1 NMR data. A restraint set co nsisting of 411 interproton distance restraints inferred from NOEs and 19 backbone and 13 side chain dihedral angle restraints from spin-spi n coupling constants, as well as 15 lower bound restraints based on th e absence of NOEs in the spectra, was used as input for distance geome try calculations in DIANA and simulated annealing and restrained energ y minimization in X-PLOR. Stereospecific assignments for 12 beta-methy lene pairs were also included. The final set of 20 structures had mean pairwise rms differences over the whole molecule of 2.04 Angstrom for the backbone heavy atoms (N, C-alpha, and C) and 2.59 Angstrom for al l heavy atoms. For the well-defined region encompassing residues 2-7 a nd 17-49, the corresponding values were 0.82 and 1.27 Angstrom, respec tively. AP-A adopts a compact structure consisting of four short stran ds of antiparallel beta-sheet (residues 2-4, 20-23, 34-37, and 45-48) connected by three loops. The first loop commences with a type I beta- turn which includes two important Asp residues; this loop is the least well-defined region of the protein, although a beta-turn involving re sidues 13-16 is observed in nearly half the structures. The loop linki ng the second and third strands is constrained by the 29-47 disulfide bond and contains two well-defined beta-turns, while the third loop co ntains the Gly40-Pro41 sequence, which has been identifed previously a s the site of cis-trans isomerism. The carboxylate group of Asp7 is cl ose to the epsilon-ammonium group of Lys37, suggesting that they may f orm a salt bridge. A pH titration monitored by 2D NMR supports this by showing that Asp7 has a low pK(a). It is proposed that this region of the molecule and the nearby residues Asp9 and His39 form part of the molecular surface which interacts with the mammalian cardiac sodium ch annel.