Neurotoxin Structure and Function

Botulinum neurotoxins are secreted from Clostridial species as a protein complex with an apparent size of approximately 900kDa.47,48 This complex consists of the neurotoxin and a number of proteins collectively called neurotoxin-associated proteins (NAPs). The NAPs include proteins classified as hemagglutinins,49,50 due to their ability to agglutinate red blood cells, and other proteins termed nontoxin nonhemagglutinins (NTNH).51,52 These other proteins stabilize the toxin and protect it from environmental degradation during passage through the gastrointestinal tract.53,54

The protein neurotoxin is secreted as a single polypeptide chain of approximately 150kDa that is nicked by proteases to form a 100-kDa heavy chain and a 50-kDa light chain connected by a single disulfide bond. The sequences of the genes encoding neurotoxin serotypes A,55-57 B,5859 C,60 D,61 E,62 f;5263 and G64 have been determined. While these toxins differ by as much as 65% at the amino acid level, it is likely that they share the same general protein fold.10 Significant sequence variability has also been observed within toxin serotypes,55,57-59 for example A1 and A2 types of neurotoxin A have been reported, which differ by 10% at the amino acid level.55,57 The differences are highest in the heavy chain. Such differences may account for the reported differences in the ability of monoclonal antibodies to recognize toxins from different A strains.65,66

Recently, the X-ray crystal structure of types A and B neurotoxins have been solved at high resolution (Figure 7.1).67-69 The structural studies, combined with functional studies, provide clear insight into how the botulinum neuro-toxins interfere with normal release of the neurotransmitter acetylcholine, resulting in flaccid paralysis (Figure 7.2). The C-terminal portion of the heavy chain (HC) comprises the receptor binding domain, which binds to cellular receptors on presynaptic neurons, resulting in toxin endocytosis70,71 (Figure 7.3). The precise determination of the cellular receptors is unknown, but the presence of two coreceptors, a protein and a sialoganglioside such as GD1b or GT1b, have been proposed.71-73 The binding domain consists structurally of an N-terminal subdomain consisting of a jelly roll motif and a C-terminal subdomain consisting of a b-trefoil motif. It is hypothesized that the C-terminal domain comprises the ganglioside binding site.67 The N-terminal portion of the heavy chain (HN) (Figure 7.1) comprises the translocation domain, which consists of a-helices and is involved in pore formation. It is hypothesized that the lower pH of the endosome induces a conformational change in this domain that creates a pore allowing the light chain to escape the endosome (Figure 7.3). The light chain (Figure 7.1) is a zinc endopeptidase which, depending on serotype, cleaves different members of the soluble -N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins,

FIGURE 7.1 Atomic structure of botulinum neurotoxin type A. Ribbon (1A) and space filling (1B) models of the X-ray crystal structure of botulinum neurotoxin type A (ref. 64). The toxin consists of a binding domain, translocation domain, and catalytic domain, as described in detail in the text.

resulting in blockade of neuromuscular transmission74'75 (Figures 7.2 and 7.3). The SNAREs are essential for normal fusion of the synaptic vesicle and acetylcholine release (Figure 7.1). Toxin serotypes A and E cleave distinct sites within SNAP-25 (synaptosomal-associated protein of 25kDa);75-78 serotypes B, D, F and G cleave distinct sites within vesicle associated membrane protein (VAMP, also known as synaptobrevin);74'75'79-82 and serotype C cleaves syntaxin and SNAP-25 (Figure 7.3).83'84 These three SNARE proteins (syntaxin, SNAP-25, and synaptobrevin) interact to form a four-helix coiled-coil in a step that precedes synaptic fusion85 (Figure 7.2). Cleavage of any one of these proteins blocks fusion and acetylcholine release, leading to a flaccid paralysis.

FIGURE 7.2 Normal neuromuscular transmission. Synaptic vesicles containing acetylcholine have the soluble -N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synap-tobrevin on their surfaces. Vesicular synaptobrevin interacts with the SNARE proteins syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) to form a four-helix coiled-coil resulting in fusion of the synaptic vesicle with the presynaptic membrane. Acetylcholine is released from the vesicle, diffuses across the synaptic cleft, and binds to the acetylcholine receptor, resulting in normal muscle contraction.

FIGURE 7.2 Normal neuromuscular transmission. Synaptic vesicles containing acetylcholine have the soluble -N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synap-tobrevin on their surfaces. Vesicular synaptobrevin interacts with the SNARE proteins syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) to form a four-helix coiled-coil resulting in fusion of the synaptic vesicle with the presynaptic membrane. Acetylcholine is released from the vesicle, diffuses across the synaptic cleft, and binds to the acetylcholine receptor, resulting in normal muscle contraction.

Light Chain Cleaves ( Synaptic V

Specific SNARE Proteins

Botulinum Toxin Endocytosed

Heavy Chain

SNARE Complex Does Not Form

Light Chain Cleaves ( Synaptic V

Specific SNARE Proteins

Botulinum Toxin Endocytosed

Heavy Chain

SNARE Complex Does Not Form

Muscle Cell Muscle Fiber Paralyzed

FIGURE 7.3 Effect of botulinum neurotoxin on normal neuromuscular transmission. Botulinum neurotoxin binds to unknown receptors on the presynaptic neuron membrane, resulting in endo-cytosis of the toxin. After endocytosis, the translocation domain changes conformation, resulting in release of the catalytic domain into the cytosol. Depending on the toxin serotype, the catalytic domain cleaves one or more members of the SNARE protein family. SNARE cleavage prevents formation of the SNARE complex and fusion of the vesicle with the membrane. As a result, acetyl-choline is not released.

Muscle Cell Muscle Fiber Paralyzed

FIGURE 7.3 Effect of botulinum neurotoxin on normal neuromuscular transmission. Botulinum neurotoxin binds to unknown receptors on the presynaptic neuron membrane, resulting in endo-cytosis of the toxin. After endocytosis, the translocation domain changes conformation, resulting in release of the catalytic domain into the cytosol. Depending on the toxin serotype, the catalytic domain cleaves one or more members of the SNARE protein family. SNARE cleavage prevents formation of the SNARE complex and fusion of the vesicle with the membrane. As a result, acetyl-choline is not released.

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