Resolving the Molecular Steps in Clostridial Neurotoxin Light Chain Translocation

Abstract

The clostridial neurotoxins (CNTs), botulinum toxin and tetanus toxin, are the most toxic proteins for humans. Neurotoxicity is based upon the specificity of the CNTs for neural host receptors and substrates. CNTs are organized into three domains, a Light Chain (LC) that is a metalloprotease and a Heavy Chain (HC) that has two domains, an N-terminal LC translocation domain (HCN) and a C-terminal receptor binding domain (HCC). While catalysis and receptor binding functions of the CNTs have been developed, our understanding of LC translocation is limited. This is due to the intrinsic complexity of the translocation process and limited tools to assess the step-by-step events in LC translocation. Recently, we developed a novel, cell-based TT-reporter to measure LC translocation as the translocation of a β-lactamase reporter across a vesicle membrane in neurons. Using this approach, we identified a role for a cis-Loop, located within the HCN, in LC translocation. In this commentary, we describe our current understanding of how CNTs mediate LC translocation and place the role of the cis-Loop in the LC translocation process relative to other independent functions that have been implicated in LC translocation. Understanding the basis for LC translocation will enhance the use of CNTs in vaccine development and as human therapies.

Figure 1:. Common routes of intoxication.

BT- and TT- routes of exposure are highlighted in yellow. Cellular mechanisms are highlighted in green and clinical presentation is highlighted in red. For BT, intestinal sporulation is observed predominantly in neonates.

Figure 2:. Mechanism of cellular intoxication.

1) At the target neuron, BT receptor binding domain (HCC) binds a synaptic vesicle protein and ganglioside receptor (peripheral motoneuron), while TT HCC binds dual gangliosides (inhibitory interneuron). 2) At peripheral motoneuron, BT enters a synaptic vesicle through receptor-mediated endocytosis, while at inhibitory interneuron, TT enters a yet to be defined vesicle. 3) As the vesicle matures, vesicular ATPases pump protons across the vesicular membrane, acidifying the lumen and inducing conformational changes in BT or TT. 4) BT and TT translocation domain (HCN) inserts into the vesicular membrane to facilitate LC translocation. 5) The interchain disulfide is reduced and the LC cleaves a SNARE-family substrate. 6) Proteolysis of the SNARE substrate inhibits SNARE-mediated vesicle fusion to the plasma membrane, uncoupling neurotransmitter release at the synaptic cleft.

Figure 3:. Structural features in Light Chain translocation.

A) Three domains of tetanus holotoxin (TT) include Light Chain (LC, pink), LC Translocation Domain (HCN, green), and Host Receptor Binding Domain (HCC, light blue). The LC-HC interchain disulfide is shown as orange spheres. Trans-end helices are highlighted in aqua, the membrane penetrating peptide (MPP) in yellow, and the viral fusion peptide (VFP) in magenta. The dashed box denotes the cis-Loop, B) BT /A (PDB 3BTA), BT/D (PDB 5BQN) , BT/E (PDB 3FFZ), and TT (PDB 5N0B) cis-Loops are rotated 180 degrees around the y axis of TT in A). The cis-loop sequence, surrounding the conserved lysine, is shown in blue.