Crystal Structure at 1.1 Å Resolution of α-Conotoxin PnIB:  Comparison with α-Conotoxins PnIA and GI

Abstract

Conotoxins are small, cysteine-rich peptides isolated from the venom of Conus spp. of predatory marine snails, which selectively target specific receptors and ion channels critical to the functioning of the neuromuscular system. alpha-Conotoxins PnIA and PnIB are both 16-residue peptides (differing in sequence at only two positions) isolated from the molluscivorous snail Conus pennaceus. In contrast to the muscle-selective alpha-conotoxin GI from Conus geographus, PnIA and PnIB block the neuronal nicotinic acetylcholine receptor (nAChR). Here, we describe the crystal structure of PnIB, solved at a resolution of 1.1 A and phased using the Shake-and-Bake direct methods program. PnIB crystals are orthorhombic and belong to the space group P212121 with the following unit cell dimensions: a = 14.6 A, b = 26.1 A, and c = 29.2 A. The final refined structure of alpha-conotoxin PnIB includes all 16 residues plus 23 solvent molecules and has an overall R-factor of 14.7% (R-free of 15.9%). The crystal structures of the alpha-conotoxins PnIB and PnIA are solved from different crystal forms, with different solvent contents. Comparison of the structures reveals them to be very similar, showing that the unique backbone and disulfide architecture is not strongly influenced by crystal lattice constraints or solvent interactions. This finding supports the notion that this structural scaffold is a rigid support for the presentation of important functional groups. The structures of PnIB and PnIA differ in their shape and surface charge distribution from that of GI.

α-Conotoxin EpI, a Novel Sulfated Peptide from Conus episcopatusThat Selectively Targets Neuronal Nicotinic Acetylcholine Receptors

“…EpI is constructed with 4 residues in loop one and 7 residues in loop two and was anticipated to have a structure similar to those reported for PnIA and PnIB (41,42). Compared with other 4/7 framework ␣-conotoxins, EpI has homology to both loop one and loop two of PnIA and PnIB and to loop one of MII (see Table I).…”

Conotoxins: Structure, Therapeutic Potential and Pharmacological Applications

“…The folding of conopeptides is mainly determined by their cysteine framework and the spacing between the cysteine residues, while the final folding of the peptide involves non-covalent interactions among non-cysteine residues [33]. However, depending upon the significant variation in spacing between the cysteines, a conserved fold may or may not be formed [42]. From the three dimensional structures known so far, conotoxins can broadly have four structural backbone folds [13].…”

“…They have a α4/7 cysteine framework with two disulfide bonds formed between Cys2 -Cys8 and Cys3 -Cys16. Their sequence comparison has shown that they differ at two positions, with Leu10 and Ser11 in PnIB replacing Ala10 and Asn11 of PnIA [42]. The overall structure of the two α-conotoxins are very similar to each other, showing a well superimposed backbone and similar side chains in most cases [42].…”

“…Their sequence comparison has shown that they differ at two positions, with Leu10 and Ser11 in PnIB replacing Ala10 and Asn11 of PnIA [42]. The overall structure of the two α-conotoxins are very similar to each other, showing a well superimposed backbone and similar side chains in most cases [42]. The largest differences observed in the main chain angles between the two α-conotoxins are ~20° for Pro13 and Tyr15.…”

A New Level of Conotoxin Diversity, a Non-native Disulfide Bond Connectivity in α-Conotoxin AuIB Reduces Structural Definition but Increases Biological Activity

“…In structures of the 4/7 ␣-conotoxins determined by x-ray crystallography, PnIA, PnIB, and EpI, a region of 3 10 helix is observed N-terminal to the ␣-helix (7,8,10). There are no solution structures reported for these molecules, but ImI has the same first loop as EpI, and four independent solution structures of ImI have been reported.…”