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The three-dimensional structure in aqueous solution of the 49-residue
polypeptide anthopleurin-A (AP-A), from the sea anemone Anthopleura
xanthogrammica, has been determined from 1H NMR data. A restraint set consisting
of 411 interproton distance restraints inferred from NOEs and 19 backbone and 13
side chain dihedral angle restraints from spin-spin coupling constants, as well
as 15 lower bound restraints based on the absence of NOEs in the spectra, was
used as input for distance geometry calculations in DIANA and simulated
annealing and restrained energy minimization in X-PLOR. Stereospecific
assignments for 12 beta-methylene pairs were also included. The final set of 20
structures had mean pairwise rms differences over the whole molecule of 2.04 A
for the backbone heavy atoms (N, C alpha, and C) and 2.59 A for all heavy atoms.
For the well-defined region encompassing residues 2-7 and 17-49, the
corresponding values were 0.82 and 1.27 A, respectively. AP-A adopts a compact
structure consisting of four short strands 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
residues 13-16 is observed in nearly half the structures. The loop linking the
second and third strands is constrained by the 29-47 disulfide bond and contains
two well-defined beta-turns, while the third loop contains the Gly40-Pro41
sequence, which has been identified previously as the site of cis-trans
isomerism. The carboxylate group of Asp7 is close to the epsilon-ammonium group
of Lys37, suggesting that they may form a salt bridge. A pH titration monitored
by 2D NMR supports this by showing that Asp7 has a low pKa. 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 channel.
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