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The clostridial neurotoxins (CNTs) comprise tetanus toxin (TT) and botulinum neurotoxin (BoNT [BT]) serotypes (A to G and X) and several recently identified CNT-like proteins, including BT/En and the mosquito BoNT-like toxin Pmp1. CNTs are produced as single proteins cleaved to a light chain (LC) and a heavy chain (HC) connected by an interchain disulfide bond. LC is a zinc metalloprotease (cleaving s oluble N -ethylmaleimide-sensitive factor a ttachment protein re ceptors [SNAREs]), while HC contains an N-terminal translocation domain (HCN) and a C-terminal receptor binding domain (HCC). HCN-mediated LC translocation is the least understood function of CNT action. Here, β-lactamase (βlac) was used as a reporter in discovery-based live-cell assays to characterize TT-mediated LC translocation. Directed mutagenesis identified a role for a charged loop ( 767 DKE 769 ) connecting α15 and α16 ( cis -loop) within HCN in LC translocation; aliphatic substitution inhibited LC translocation but not other toxin functions such as cell binding, intracellular trafficking, or HCN-mediated pore formation. K 768 was conserved among the CNTs. In molecular simulations of the HCN with a membrane, the cis -loop did not bind with the cell membrane. Taken together, the results of these studies implicate the cis -loop in LC translocation, independently of pore formation. IMPORTANCE How protein toxins translocate their catalytic domain across a cell membrane is the least understood step in toxin action. This study utilized a reporter, β-lactamase, that was genetically fused to full-length, nontoxic tetanus toxin (βlac-TT) in discovery-based live-cell assays to study LC translocation. Directed mutagenesis identified a role for K 768 in LC translocation. K 768 was located between α15 and α16 (termed the cis -loop). Cellular assays showed that K 768 did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K 768 is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis -loop appears to contribute to LC translocation.

Tetanus Toxin cis -Loop Contributes to Light-Chain Translocation