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Membrane-induced refolding of tetanus neurotoxin translocation domain
O'Neil, Pierce
O'Neil, Pierce
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Abstract
The bacterium Clostridium tetani causes the disease tetanus, which is characterized by spastic paralysis. Paralysis is caused by a secreted, soluble protein that interferes with normal neuronal function. Based upon its neurotoxicity, the identified protein was named tetanus neurotoxin (TeNT). With the identification of the protein, studies into its structure and function have shown that TeNT is a member of the A-B toxin family. All A-B toxins have an active (A), or enzymatic, subunit and a binding (B) subunit. For TeNT, the B subunit can be divided into two functional domains: the translocation (HCT) domain and the receptor binding domain. The receptor binding domain binds neuron-specific protein and lipid receptors. The translocation domain, when triggered by endosomal acidification, interacts with and inserts into the membrane. The latter step is accompanied by the translocation of the A subunit across the endosomal membrane. Once inside a target cell, the A subunit, a zinc endopeptidase, disrupts neurotransmitter release from affected neurons, which is the direct cause of paralysis. In addition to translocation, HCT forms a final membrane-inserted state that is cation conductive. Although the function of HCT is well defined, the mechanism by which it accomplishes this task remains unknown. As part of the mechanism, HCT has been hypothesized to undergo extensive structural rearrangement upon interaction with membranes. To gain insight into the HCT-membrane interaction, we evaluated the structural features of (i) the final, membrane-inserted state of truncated HCT (“bHCT”) and (ii) the formation of a pre-insertion, membrane-competent state in full-length, soluble TeNT. Results demonstrated that the acid- and membrane-induced refolding of TeNT bHCT culminated with the majority of this domain being inserted into the membrane. The soluble domain comprised primarily alpha helices that refolded upon insertion to comprise alpha-helices distributed both interfacially (near the membrane surface) and embedded in the hydrocarbon core (near the center of the bilayer). Although the presence of transmembrane helices was confirmed, the helical boundaries could neither be established experimentally nor predicted computationally. Thus, one possible outcome is that the final, membrane-inserted state disrupts the normal membrane structure in a manner similar to some viral proteins. In relation to other A-B toxins, our findings for TeNT were not similar to those of the anthrax toxin pore, which forms a stable, beta-barrel structure that minimally inserts into membranes. Instead, our findings for TeNT were more similar to studies of diphtheria toxin, which forms a cation conductive structure involving alpha-helices and insertion of the majority of the analogous domain. Unlike diphtheria toxin, however, TeNT bHCT lacked clear, helical boundaries. Indeed, although TeNT may be more similar to diphtheria toxin than anthrax, TeNT may represent a novel membrane-inserted structure and mechanism. In addition to our bHCT results, we performed a preliminary study to show that full-length, soluble TeNT, at neutral pH, has an exposed hydrophobic region, which may reflect a pre-insertion conformational change that is required for insertion. By further understanding the mechanism by which TeNT HCT interacts membranes, this work expands the current literature of protein-membrane interaction and for the first time provides evidence for the structure of the final membrane-inserted TeNT state.
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2021-01-01
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University of Kansas
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Keywords
Biochemistry, Acid-induced Protein-lipid Interactions, Biolayer Interferometry, Fluorescence, Protein-membrane Interaction, Tetanus neurotoxin