|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.
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.
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 KM (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.
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 KM 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).