Mechanism of botulinum neurotoxin transport across membranes

PROJECT SUMMARYBotulinum neurotoxins (BoNTs) are among the most toxic agents known to humans and cause the lifethreatening, neuroparalytic disorder botulism. The potential for major public health impact resulting for anintentional release, combined with the paucity of approved vaccines or therapies has led to the classification ofBoNTs as Tier 1, Category A Select Agents. Paradoxically, the highly specific action of BoNTs make themexcellent therapeutics for a growing and heterogeneous number of human diseases that are characterized by ahyperactivity of peripheral nerve terminals. Despite many recent advances in understanding the structure-function relationship of BoNTs, the molecular events by which the neurotoxin heavy chain (HC) is able totranslocate the light chain (LC) across the membrane of endocytic vesicles remains poorly defined.Understanding the mechanism of pH-driven neurotoxin unfolding and translocation is not only of intrinsic value,but also addresses general biophysical questions underlying membrane protein assembly and stability. Site-selective fluorescence labeling of neurotoxins in conjunction with an array of biochemical, spectroscopic andmolecular approaches will be employed to test the central hypothesis stating that membrane insertion of BoNTis a regulated process containing key intermediate states which precede formation of the protein translocatingchannel. Aim 1 will determine the contribution of the receptor binding (HCR) domain in formation of themembrane inserted channel. Specifically, aim 1 will test the hypothesis that the HCR domain functions as asensor of environmental pH and membrane composition which ensures channel formation occurs at the correctsite and time. Aim 2 will address the hypothesis that formation of the BoNT/A channel occurs through a seriesof interfacial intermediate states. Completion of the proposed studies will provide opportunities for thedevelopment of post-exposure therapeutics and improved pharmacologic agents for the treatment of neuronaldisorders.