Title: Cloning, expression and purification of botulinum neurotoxin type A heavy chain - crystallographic evidence for a putative tetrameric pore

Authors: Damodharan Lakshminarasimhan; Desigan Kumaran; Rakhi Agarwal; Bal Ram Singh; Subramanyam Swaminathan

Addresses: Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA. ' Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA. ' Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA. ' University of Massachusetts Dartmouth, Dartmouth, MA 02747, USA ' Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.

Abstract: Clostridium botulinum produces seven antigenically distinct serotypes of botulinum neurotoxin (BoNTs, A-G) the most potent toxins to humans. They cause paralysis at less than picomolar concentration by blocking neurotransmitter release. BoNT consists of a heavy chain and a light chain. The C-terminal half of the heavy chain allows the toxin to bind to the presynaptic membrane while the N-terminal half helps in forming a channel in the endosomal membrane to allow the light chain to escape into the cytosol. While the binding and catalytic mechanisms are well understood now, the details of translocation still remain a mystery. A full length BoNT/A heavy chain (BAHC) has been cloned, over expressed and purified from inclusion bodies by solubilising with a detergent. Preliminary crystallographic results show that BAHC forms a tetramer in the crystal lending experimental support for tetrameric pore formation for the light chain to pass through the endosomal membrane.

Keywords: botulinum neurotoxins; BoNTs; translocation; oligomerisation; pore formation; heavy chains; auto induction; inclusion bodies; crystallography; tetrameric pores; botulism; clostridium botulinum; endosomal membrane.

DOI: 10.1504/TBJ.2012.050196

The Botulinum Journal, 2012 Vol.2 No.2, pp.135 - 149

Published online: 30 Oct 2014 *