Botulinum Neurotoxin Devoid of Receptor Binding Domain Translocates Active Protease

Abstract

Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The ∼50 kDa light chain (LC) protease is translocated into the cytosol by the ∼100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication.

Tetanus Neurotoxin Utilizes Two Sequential Membrane Interactions for Channel Formation

“…Therefore, what role, if any, does lipid composition play in channel formation by BoNTs? Interestingly, channel formation in planar bilayer systems employed either asolectin or defined lipid mixtures containing both phosphatidylserine and ganglioside GT1b (21,43). Thus, the requirement for acidic lipids in regulating the formation of the TeNT interfacial intermediate state may be a shared property among the CNTs.…”

“…Thus, the requirement for acidic lipids in regulating the formation of the TeNT interfacial intermediate state may be a shared property among the CNTs. Indeed, Fischer et al (21) previously noted that translocation activity of BoNT/A devoid of the receptor binding domain could not be observed in non-neuronal cell lines and speculated that membrane lipid composition might contribute to this effect.…”

“…Traditionally low pH was proposed to trigger the translocation process, presumably by promoting structural changes facilitating the insertion of the HCT into the membrane bilayer. However, the recent demonstration that the isolated HCT of BoNT/A can form ion-conducting channels in the absence of a transmembrane pH gradient brings this model into question (20,21). Rather, it appears that low pH serves to (i) relieve the inhibition of the translocation process mediated by the HCR and (ii) facilitate the partial unfolding of the LC into a conformation necessary for passage through the translocation channel (22,23).…”

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

“…It was found that BoNT/A, in fact, possesses SNP-binding regions on both the H N and the H C domains (20,27). Employing dual detection of substrate proteolysis and single-channel currents, Fischer et al (21) showed that a protein consisting only of the L chain and the H N domain enabled passage of active protease into the cytosol of target cells. The H C domain appeared to be unnecessary for cell entry, channel action, or L-chain translocation, which suggested that each unit chaperones the others to attain toxicity (44).…”

“…Our previous studies (20) using short (19-residue) overlapping peptides to map the synaptosome (SNP) binding regions on the H chain of BoNT/A showed that certain regions of the H N domain as well as the H C domain are capable of inhibiting BoNT binding to SNPs. Another study (21) reported that the L chain can be translocated efficiently by the H N domain alone in the absence of the H C domain. However, the precise role of the H N domain in membrane binding and insertion or channel formation is still unknown (22).…”