Abstract
SmIIIA is a new micro-conotoxin isolated recently from Conus stercusmuscarum. Although it shares several biochemical characteristics with other micro-conotoxins (the arrangement of cysteine residues and a conserved arginine believed to interact with residues near the channel pore), it has several distinctive features, including the absence of hydroxyproline, and is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be characterized. It therefore represents a potentially useful tool to investigate the functional roles of these channels. We have determined the three-dimensional structure of SmIIIA in aqueous solution. Consistent with the absence of hydroxyprolines, SmIIIA adopts a single conformation with all peptide bonds in the trans configuration. The spatial orientations of several conserved Arg and Lys side chains, including Arg14 (using a consensus numbering system), which plays a key role in sodium channel binding, are similar to those in other micro-conotoxins but the N-terminal regions differ, reflecting the trans conformation for the peptide bond preceding residue 8 in SmIIIA, as opposed to the cis conformation in micro-conotoxins GIIIA and GIIIB. Comparison of the surfaces of SmIIIA with other micro-conotoxins suggests that the affinity of SmIIIA for TTX-resistant channels is influenced by the Trp15 side chain, which is unique to SmIIIA. Arg17, which replaces Lys in the other micro-conotoxins, may also be important. Consistent with these inferences from the structure, assays of two chimeras of SmIIIA and PIIIA in which their N- and C-terminal halves were recombined, indicated that residues in the C-terminal half of SmIIIA confer affinity for tetrodotoxin-resistant sodium channels in the cell bodies of frog sympathetic neurons. SmIIIA and the chimera possessing the C-terminal half of SmIIIA also inhibit tetrodotoxin-resistant sodium channels in the postganglionic axons of sympathetic neurons, as indicated by their inhibition of C-neuron compound action potentials that persist in the presence of tetrodotoxin.
Highlights
SmIIIA is a new -conotoxin isolated recently from Conus stercusmuscarum
The spatial orientations of several conserved Arg and Lys side chains, including Arg14, which plays a key role in sodium channel binding, are similar to those in other -conotoxins but the N-terminal regions differ, reflecting the trans conformation for the peptide bond preceding residue 8 in SmIIIA, as opposed to the cis conformation in -conotoxins GIIIA and GIIIB
Comparison of the surfaces of SmIIIA with other -conotoxins suggests that the affinity of SmIIIA for TTX-resistant channels is influenced by the Trp15 side chain, which is unique to SmIIIA
Summary
Peptide Synthesis and Sample Preparation—Conotoxins SmIIIA and PIIIA and chimeras were synthesized, folded, and purified using the protocols described previously [12, 14]. Structure Calculations—Two sets of structural calculations were performed in CYANA [24] Both used a set of four peak lists derived from the NOESY spectra described above. Intensities for each peak were calibrated using the conventional CALIBA script supplied with CYANA These were scaled by a factor of 1.2 before use in structural calculations to allow for possible effects of spin diffusion. The peak lists used for the manual assignments were supplied to CANDID and NOE assignments and structures were determined using the default scripts. The final NOE and dihedral restraint lists from CYANA were used to calculate a family of 200 structures in Xplor-NIH [25] using the standard distance geometry and simulated annealing scripts.
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