Abstract
Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC.
Highlights
Botulinum neurotoxin (BoNT) secreted by Clostridium botulinum is the causative agent of botulism, a neuroparalytic disease
Three protein constructs were used in this study: LC and HN, corresponding to the isolated catalytic and translocation domains of Botulinum neurotoxin A (BoNT/A), and LC-HN, corresponding to a truncated BoNT/ A toxin lacking the HC domain (Fig 1)
Analysis of the secondary structure of the three proteins from these Circular dichroïsm (CD) data is in agreement with the crystal structure of BoNT/A (Fig 1 and Table 1) [4]
Summary
Botulinum neurotoxin (BoNT) secreted by Clostridium botulinum is the causative agent of botulism, a neuroparalytic disease. There are seven serologically distinct BoNT isoforms designated as A to G [1] These toxins are considered to be the most potent among bacterial, animal, plant toxins and chemical compounds. Membrane Interaction of C and T Domains of BoNT/A botulism, BoNTs are considered as a potential biological weapon [2]. It is a remarkable cure for numerous neuromuscular and neuronal diseases [3]. The heavy chain (HC, 100 kDa) encompasses two functional domains: the translocation domain at the N-terminus (HN) and the receptor-binding domain at the C-terminus (HC) [4,6,7]. LC cleaves SNARE proteins involved in neurotransmitter release leading to the inhibition of acetylcholine release at the neuromuscular junction, resulting in flaccid paralysis [16]
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