Faculty Collaboration Database

Tetanus Toxin cis-Loop Contributes to Light-Chain Translocation. mSphere 2020 May 06;5(3)

Date

05/08/2020

Pubmed ID

32376703

Pubmed Central ID

PMC7203457

DOI

10.1128/mSphere.00244-20

Scopus ID

2-s2.0-85084398090 (requires institutional sign-in at Scopus site)   8 Citations

Abstract

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 soluble N-ethylmaleimide-sensitive factor attachment protein receptors [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 (⁷⁶⁷DKE⁷⁶⁹) 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. K⁷⁶⁸ 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 K⁷⁶⁸ in LC translocation. K⁷⁶⁸ was located between α15 and α16 (termed the cis-loop). Cellular assays showed that K⁷⁶⁸ did not interfere with other toxin functions, including cell binding, intracellular trafficking, and pore formation. The equivalent K⁷⁶⁸ is conserved among the clostridial neurotoxin family of proteins as a conserved structural motif. The cis-loop appears to contribute to LC translocation.

Author List

Zuverink M, Bluma M, Barbieri JT

Author

Joseph T. Barbieri PhD Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Botulinum Toxins
Cell Line
Cell Membrane
Cells, Cultured
Mice
Neurons
Protein Binding
Protein Transport
Rats
Tetanus Toxin
Translocation, Genetic