The long-term goal of this project is the elucidation of the molecular mechanisms by which the snake-venom derived, polypeptide alpha -neurotoxins block nicotinic acetylcholine receptor (nAChR) function. We will focus on the prototypical long-chain alpha -neurotoxin, alpha -bungarotoxin (Bgtx), the """"""""gold standard"""""""" of alpha -neurotoxins. Bgtx has been used extensively in the study of muscle-type nAChRs and a subset of neuronal nAChRs sensitive to Bgtx inhibition (e.g., alpha -7containing nAChRs). The questions that we intend to address are: 1.) Which specific residues and general structural and dynamic features in Bgtx are important for recognition and block of Bgtx-sensitive nAChRs? 2.) Can the receptor specificity of Bgtx be altered through mutagenesis to produce """"""""gain of function"""""""" variants capable of interacting in a selective manner with neuronal nAChRs that are normally insensitive to Bgtx? and 3.) How do Bgtx mutations that disrupt or significantly alter toxin activity affect the structure and conformational flexibility of Bgtx? We will pursue a structure-function analysis of Bgtx, utilizing site-directed mutagenesis of recombinant Bgtx produced in Pichia pastoris and double mutant cycle analysis to identify sites responsible for the high-affinity neuromuscular blockade produced by this alpha -neurotoxin. We also intend to identify and characterize """"""""gain of function"""""""" mutations of Bgtx which enable the recognition and functional inhibition of neuronal nAChRs that are otherwise insensitive to blockade by native Bgtx. This approach should lead to a new battery of toxins that differentially bind to the various neuronal nAChR subunit combinations. We plan to determine the structure of the recombinant Bgtx by multidimensional NMR, to compare this structure to the NMR solution structure of native Bgtx, and to analyze the backbone dynamics of recombinant Bgtx metabolically labeled with15N. Labeled Bgtx would also allow a characterization of atomic motions within its various functionally important regions. Local confirmational flexibility in Bgtx may be an important factor in the mechanism of Bgtx/receptor recognition and binding and could very well contribute both to the high affinity of Bgtx and to its ability to bind to a large number of nAChRs of varying primary structure. The proposed studies promise to shed light on the fundamental mechanisms by which protein alpha -neurotoxins interact tightly and specifically with their target sites on the cell surface. This information will be useful in providing valuable models for drug design efforts and in developing new pharmacological tools for the study of neuronal nAChRs.
