Tetanus neurotoxin utilizes two sequential membrane interactions for channel formation.

Tetanus neurotoxin (TeNT) causes neuroparalytic disease by entering the neuronal soma to block the release of neurotransmitters. However, the mechanism by which TeNT translocates its enzymatic domain (light chain) across endosomal membranes remains unclear. We found that TeNT and a truncated protein devoid of the receptor binding domain (TeNT-LHN) associated with membranes enriched in acidic phospholipids in a pH-dependent manner. Thus, in contrast to diphtheria toxin, the formation of a membrane-competent state of TeNT requires the membrane interface and is modulated by the bilayer composition. Channel formation is further enhanced by tethering of TeNT to the membrane through ganglioside co-receptors prior to acidification. Thus, TeNT channel formation can be resolved into two sequential steps: 1) interaction of the receptor binding domain (heavy chain receptor binding domain) with ganglioside co-receptors orients the translocation domain (heavy chain translocation domain) as the lumen of the endosome is acidified and 2) low pH, in conjunction with acidic lipids within the membrane drives the conformational changes in TeNT necessary for channel formation.