Membrane Interaction of Botulinum Neurotoxin A Translocation (T) Domain

Abstract

The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.

Studies of the Mechanistic Details of the pH-dependent Association of Botulinum Neurotoxin with Membranes

“…It is thought that the acidic pH of the endosome triggers HCT pore formation and LC translocation (6). In vitro studies have shown that BoNT (and the isolated BoNT HCT) undergoes pH-dependent membrane insertion and pore formation (7)(8)(9)(10)(11)(12)(13), and single molecule translocation events have been observed in excised patches of neuronal cells (14,15). These studies support a model in which the HCT acts as both a conduit and a chaperone for the transit of the LC protease across the membrane (5,11,16).…”

The C-Terminal Heavy-Chain Domain of Botulinum Neurotoxin A Is Not the Only Site That Binds Neurons, as the N-Terminal Heavy-Chain Domain Also Plays a Very Active Role in Toxin-Cell Binding and Interactions

“…Cardiolipin has two phosphate groups, but it effectively behaves like it has one negative charge at physiological pH. It appears that the binding of H N 729 -845 is primarily aided by the negative charge on the head of the gangliosides or the lipid and is consistent with the findings of Galloux et al (48), where the membrane surface charges are reported to play an active role in the H N domain-membrane interaction. Involvement of the surface charge in interactions of H N 729 -845 with the cell explains the binding of the peptide to CHO-K1 and HEK-293T cells.…”

“…This was further supported by reports where the H N domain-L-chain framework was used as a secretion inhibitor by fusion to a new cell-targeting domain (45)(46)(47). Using physicochemical and spectroscopic methods, Galloux et al (48) found that the H N domain interacts with the membrane; however, the interaction did not involve major secondary or tertiary structural changes. Below its pI of 5.5, the H N domain becomes insoluble and is capable of penetrating lipid vesicles.…”

Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons

“…The identity of the protein receptor for BoNT/C remains to be established. Upon SV endocytosis, the drop in luminal pH triggers the transformation of the HC into a translocation machine (Fischer, 2013, Montal, 2010, Williamson and Neale, 1994, Fu et al, 2002, Puhar et al, 2004, Galloux et al, 2008, Pirazzini et al, 2013); interestingly, the ability to sense low pH requires the interaction of the HC with the ganglioside co-receptor (Sun et al, 2011). The HC then translocates the LC into the cytosol, where it cleaves neuronal soluble N- ethylmaleimide attachment receptors (SNAREs), which form the core of a conserved membrane fusion complex (Rothman, 1994).…”