Structural basis for alpha-conotoxin potency and selectivity

Abstract

Parkinson's disease is a debilitating movement disorder characterized by altered levels of alpha(6)beta(2) * ( * indicates the possible presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) localized on presynaptic striatal catecholaminergic neurons. alpha-Conotoxin MII (alpha-CTx MII) is a highly useful ligand to probe alpha(6)beta(2) nAChRs structure and function, but it does not discriminate among closely related alpha(6) * nAChR subtypes. Modification of the alpha-CTx MII primary sequence led to the identification of alpha-CTx MII[E11A], an analog with 500-5300-fold discrimination between alpha(6) * subtypes found in both human and non-human primates. alpha-CTx MII[E11A] binds most strongly (femtomolar dissociation constant) to the high affinity alpha(6) nAChR, a subtype that is selectively lost in Parkinson's disease. Here, we present the three-dimensional solution structure for alpha-CTx MII[E11A] as determined by two-dimensional (1)H NMR spectroscopy to 0.13+/-0.09A backbone and 0.45+/-0.08A heavy atom root-mean-square deviation from mean structure. Structural comparisons suggest that the increased hydrophobic area of alpha-CTx MII[E11A] relative to other members of the alpha-CTx family may be responsible for its exceptionally high affinity for alpha6alpha4beta2 * nAChR as well as discrimination between alpha(6)beta(2) and alpha(3)beta(2) containing nAChRs. This finding may enable the rational design of novel peptide analogs that demonstrate enhanced specificity for alpha(6) * nAChR subunit interfaces and provide a means to better understand nAChR structural determinants that modulate brain dopamine levels and the pathophysiology of Parkinson's disease.

Figure 1

A structure comparison of α-CTxs MII (top row) and E11A (bottom row). From left to right, an overlay of the 20 backbone atoms for the final 20 structures (for MII, Shon et al. structure) with the N- and C-termini labeled, a tube structure with Pro⁶ labeled as the point of initiation of the α-helix and residues in positions 11 and 12 labeled, and lastly, the surface of each peptide colored by atom charge where red is negative, blue is positive, and white is neutral for the peptides at a pH of 3.

Figure 2

Surface comparison of α-CTxs PIA, MII[E11A], BuIA, MII, GIC, OmIA from top to bottom, respectively. Blue regions represent electropositive surfaces, red regions are electronegative surfaces, and white/faintly colored regions are hydrophobic/nonpolar surfaces. Sulfur atoms of cystine residues are yellow. Left panel: front view of peptide surface. Right panel: backside view of peptide surface.