Molecular engineering of conotoxins:: The importance of loop size to α-conotoxin structure and function

alpha-Conotoxins are competitive antagonists of nicotinic acetylcholine receptors (nAChRs). The majority of currently characterized alpha-conotoxins have a 4/7 loop size, and the major features of neuronal alpha-conotoxins include a globular disulfide connectivity and a helical structure centered around the third of their four cysteine residues. In this study, a novel "molecular pruning" approach was undertaken to define the relationship between loop size, structure, and function of a-conotoxins. This involved the systematic truncation of the second loop in the a-conotoxin [A10L]PnIA [4/7], a potent antagonist of the alpha 7 nAChR. The penalty for truncation was found to be decreased conformational stability and increased susceptibility to disulfide bond scrambling. Truncation down to 4/4[A10L]PnIA maintained helicity and did not significantly reduce electrophysiological activity at alpha 7 nAChRs, whereas 4/3[AIOL]PnIA lost both alpha 7 nAChR activity and helicity. In contrast, all truncated analogues lost similar to 100-fold affinity at the AMP, a model protein for the extracellular domain of the nAChR. Docking simulations identified several hydrogen bonds lost upon truncation that provide an explanation for the reduced affinities observed at the alpha 7 nAChR and AChBP.