Botulinum Neurotoxin is Bio-shielded by NTNHA in a Handshake Complex
SSRL Science
Summary - October 2012
Botulinum neurotoxins (BoNTs) invade motor neurons at their junctions with
muscular tissue, where the toxins disable the release of the neurotransmitter
acetylcholine and subsequently paralyze the affected muscles. Accidental BoNT
poisoning primarily occurs through ingestion of food products contaminated by
Clostridium botulinum, the bacterium that produces BoNTs. However,
BoNTs by
themselves are fragile and sensitive to low pH environments and digestive
proteases. So how do they survive the harsh environment of the host's
gastrointestinal tract?
Researchers at Sanford-Burnham Research Institute and the Medical School of
Hannover in Germany recently discovered that the neurotoxin has a bodyguard, a
protein termed non-toxic non-hemagglutinin (NTNHA) protein that is produced by
the C. botulinum bacterium simultaneously with BoNT. This study,
published in Science, revealed the first crystal structure of
a ~300kDa protein complex composed of serotype A BoNT (BoNT/A) and NTNHA, which
together form the minimally functional progenitor toxin complex (M-PTC) (Figure
1). A genetically modified, catalytically inactive BoNT/A that carries three
mutations in its catalytic site (termed BoNT/Ai) was used for the structural
studies. The M-PTC crystal structure was determined at 2.7 Å resolution
by single isomorphous replacement with anomalous scattering (SIRAS) method
using x-ray diffraction data collected at SSRL's Beam Line 9-2 and NE-CAT at
the Advanced Photon Source. Surprisingly, the non-toxic NTNHA exhibited a
three-domain architecture similar to the neurotoxin, although the two proteins
share less than 20% sequence identity (Figure 1). The two proteins hug each
other, interlocking with what looks like a handshake and providing unusually
large and multivalent binding interfaces that shield BoNT from the hostile
gastrointestinal environment. Biochemical and functional studies also revealed
a novel pH-sensing mechanism which enables the M-PTC to remain tightly bound at
acidic pH in the gut, dissociating at neutral pH after absorption from the gut
into the general circulation.
Several essential residues involved in pH-dependent assembly of BoNT/A and
NTNHA, which are weak spots of the toxin that could be targeted with new
therapeutics, were identified by structure-based mutagenesis. This discovery
suggests an innovative approach for oral inhibitor design that aims to mimic pH
change by prematurely breaking up BoNT's protective embrace and leaving the
unprotected toxin to be targeted by the stomach's digestive enzymes and acid.
This type of therapy-either alone or in combination with other therapies
currently in development-could be given preventively at a time when BoNT
contamination becomes a public health concern. Furthermore, a better
understanding of the toxin protection mechanism could enable the design of
novel proteinaceous vehicles to allow oral administration of drugs that
currently must be injected.
Primary Citation
Shenyan Gu, Sophie Rumpel, Jie Zhou, Jasmin Strotmeier, Hans Bigalke, Kay Perry, Greice Krautz-Peterson, Charles B. Shoemaker, Andreas Rummel, Rongsheng Jin. (2012) "Botulinum neurotoxin is shielded by NTNHA in an interlock complex." Science, 335 (6071): 977-981. [DOI: 10.1126/science.1214270]Related Links
Contact
Rongsheng Jin, The Sanford-Burnham Research Institute
