Crystal Structure Studies Of α-Conotoxins In Complex With Acetylcholine-binding Proteins

The a-conotoxins are the most studied conotoxins currently because they inhibit nicotiniacetylcholine receptors (nAChRs) at nerves and muscles. As the nAChR antagonists, a-conotoxins have exhibited remarkable neuropharmacological effects in the treatment for pain, Alzheimer’s disease, epilepsy and addiction to smoking etc. The acetylcholine binding proteins (AChBPs) are homologous to the ligand-binding domains of the nAChRs and also have similar neuropharmacological characteristics. Therefore, solving the crystal structures of AChBPs in complex with a-conotoxins will provide us more insights into the functions of a-conotoxins in physiology and pharmacology, and also help us to discover and design a-conotoxin-based drugs.In this study, we have used Bac-to-Bac baculovirus system to efficiently expresse Ac-AChBP protein for co-crystalization with several synthezed a-conotoxin peptides, such as GIC. The crystal robot was employed to optimize crystal growth conditions, and the best buffer compostions found for this co-crystalization was0.6M lithium sulfate monohydrate with0.1M sodium acetate trihydrate pH4.6. The complex crystals of Ac-AChBP with a-conotoxin GIC were successfully obtained and further applied for X-ray structural studies. We have finally solved the co-structures of acetylcholine binding protein (AChBP) with the a-conotoxin GIC at a high resolution of2.4A. Using the X-ray structures, we have analyzed the more detailed contacts of a-conotoxin GIC residues interacting with Ac-AChBP residues. The GIC H5was found to interact with Gln184and Tyr186of the loop C principal site. On the complementary site of Ac-AChBP, Q13of GIC positioned into a hydrophobic pocket that was formed by Va1106, Thr108, Ser112and Met114of Ac-AChBP. The a-conotoxin Q13serves as an anchoring point to confer specificity and affinity for the binding.Based on the co-structures and the molecular docking, we have also discovered that H5, Q13of a-conotoxin GIC were the key residues for α3β2nAChR highly selectivity, but not for α4β2or α3β4nAChR. So this study has also revealed the ion channels or receptors subtype selections for the binding of particular a-conotoxins, and this information will be helpful for the design of new analog drugs of a-conotoxins for different nAChRs.