Substrate Recognition Strategy for Botulinum Neurotoxin

Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. By inhibiting release of the neurotransmitter acetylcholine, for example, the neurotoxin produced by the bacterium Clostridium botulinum interferes with nerve impulses and causes a paralysis of respiratory and skeletal muscles that can cause death. Researchers from Stanford University have now determined the first structure of a CNT in complex with its target. The structure at a resolution of 2.1 Å, together with enzyme kinetic data, reveals an array of active sites (exosites) that give the CNT its deadly specificity.

Botulism Toxin

The best thing one can say about botulism is that it is relatively rare. At worst, botulism can ultimately result in death due to respiratory failure resulting from paralysis of the muscles used in breathing. The muscle paralysis is caused by the neurotoxin produced by the bacterium Clostridium botulinum, which can be ingested with contaminated food, enter the body through wounds, or (in the case of infants where there is little competition from other microbes) germinate in the intestine from spores. Given early after onset of symptoms, an antitoxin exists that can prevent further damage, but a more effective treatment for this and other neurotoxin-caused illnesses, such as tetanus, await a more detailed understanding of how the toxin works at the molecular level.

Breidenbach and Brunger have taken a step toward toward this goal with the first structure, obtained by protein (x-ray) crystallography, of a key component of the botulinum neurotoxin while bound to its target molecule. It was already known that the target, known as SNAP-25, is required for the release of the neurotransmitter needed for normal function; however, when SNAP-25 is cleaved, the release of neurotransmitter is inhibited. The new structure allowed the team to generate a detailed model of how the binding between the botulinum neurotoxin component and SNAP-25 that leads to cleavage comes about. The model may lead to the development of inhibiting agents that prevent the binding.

CNTs contain enzymes that do their damage by impairing neuronal exocytosis (the process by which neurotransmitter is released into a synapse). In normal function, it is believed that assembly of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) into a low-energy ternary complex catalyzes membrane fusion, which precipitates the neurotransmitter release.

CNTs interfere with this process by degrading SNAREs, a process called proteolysis. Toxins like botulinum neurotoxins are two-chain polypeptides with a heavy chain joined by a disulphide bond to a light chain. CNT light chains are part of a unique group of zinc-dependent endopeptidases (enzymes) that catalyze hydrolysis of peptide bonds within other proteins. When such chains catalyze SNARE hydrolysis at specific sites, membrane fusion is attenuated and neurotransmitter release inhibited.

Three different views of the complex between SNAP-25 (red) and BoNT/A (tan). The views are related by the specified rotations around a vertical axis in the plane of the figure that goes through the center of the complex. Shown are the α exosite (green arrow and circle), BoNT/A light-chain helices (tan) α1–α4, the helical N-terminal of the substrate (red), approximate locations (green) of contacting side chains involved in the α exosite, the β exosite (blue arrow and circle), the “250 loop” (light blue), the active site (Zn²⁺, purple sphere), the “370 loop” (light blue), approximate locations (dark blue) of contacting side chains involved in the β exosite, and the approximate locations (yellow) of other exosites (anchor points) involved in side-chain contacts.

The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation. It is thought to use one or more regions of enzyme–substrate interaction (exosites) remote from the active site where hydrolysis occurs. To address this question, the Stanford team used x-ray diffraction data from ALS Beamlines 8.2.1 and 8.2.2 and SSRL Beamline 9-2 to determine the first structure of the light chains of a CNT endopeptidase (the protease botulinum neurotoxin serotype A or BoNT/A) in complex with its target SNARE (human SNAP-25).

In their structure, an α exosite is formed by BoNT/A light-chain helices α1–α4 that bind to the helical N-terminal of the substrate SNAP-25. Contacting side chains between the SNAP-25 substrate and the BoNT/A light chain at the α exosite are located along the helices. A β exosite resides on the opposite face of BoNT/A. The C-terminal of SNAP-25 forms an antiparallel β sheet along with a portion of a “250 loop,” which is separated from the zinc-containing active site where substrate cleavage occurs by a “370 loop.” Additional exosites (anchor points) also include side-chain contacts between the substrate and the light chain.

Based on their structure and available kinetic data for several mutant SNAP-25 substrates, the researchers concluded that most of this unusually large enzyme–substrate interface serves to provide a substrate-specific boost to catalytic efficiency by reducing K_M (the Michaelis constant), so that the substrate is properly oriented with respect to the light chain. They also observed significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis.

Exosite-based model of BoNT/A substrate recognition. Left: SNAP-25 attaches to a presynaptic membrane via palmitylation sites (black) on its linker domain (purple). Center: Helix formation at the α exosite probably initiates binding of BoNT/A (blue) to the SNAP-25 C-terminal domain (green), and the anchor points along the domain (green notches) increase substrate specificity. Right: The resulting enhanced binding at the β exosite induces conformational changes at the active site (AS) that allow cleavage of the SNAP-25.

According to a general model of the strategy used by BoNT/A to recognize and cleave SNAP-25 based on this structure, SNAP-25 attaches to a presynaptic membrane via palmitylation (fatty acid binding) sites on its linker domain. The N-terminal (sn1) and C-terminal (sn2) domains are unstructured or flexible in uncomplexed SNAP-25. Binding of BoNT/A is probably initiated by helix formation at the α exosite, and the anchor points along the extended portion of SNAP-25 serve as additional determinants of substrate specificity. These sites reduce K_M and enhance binding at the β exosite, thereby inducing conformational changes at the zinc active site that render the endopeptidase competent to cleave its substrate.

Ultimately, the novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.

Research conducted by M.A. Breidenbach (Stanford University) and A.T. Brunger (Howard Hughes Medical Institute, Stanford University, and Stanford Synchrotron Radiation Laboratory).

Research funding: National Institutes of Health and Howard Hughes Medical Institute. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: M.A. Breidenbach and A.T. Brunger, “Substrate recognition strategy for botulinum neurotoxin serotype A,” Nature 432, 925 (2004).