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Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity.

Xue S, Javor S, Hixon MS, Janda KD - Biochemistry (2014)

Bottom Line: Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity.A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors.These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo.

View Article: PubMed Central - PubMed

Affiliation: Departments of Chemistry and Immunology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

ABSTRACT
Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an α-exosite binding probe. Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo.

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Structure of BoNT/A-LC (424 a.a. resolved, green)and SNAP-25 fragment(64 a.a. C-terminal, 59 a.a. resolved). BAP-24 region is shown inred, while the remaining SNAP-25 residues are shown in cyan and theα-exosite in blue (PDB 1XTG).
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fig1: Structure of BoNT/A-LC (424 a.a. resolved, green)and SNAP-25 fragment(64 a.a. C-terminal, 59 a.a. resolved). BAP-24 region is shown inred, while the remaining SNAP-25 residues are shown in cyan and theα-exosite in blue (PDB 1XTG).

Mentions: To explore how α-exosite binding influences the activityof BoNT/A LC, a probe was designed based on the published crystalstructure of BoNT/A LC complexed with SNAP-25 (Figure 1, PDB 1XTG).6 The probe consisted of the helicalportion of SNAP-25 that binds to the α-exosite, spanning 24amino acids, which we term BAP-24 (BoNT/A LC α-exosite probe24). Additionally, within BAP-24, we highlight that the C-terminalalanine was replaced by aminoisobutyric acid, a well studied effectiveinducer of α-helical structure,17 which assists BAP-24 to adopt a helical structure (Figure 2). As anticipated, BAP-24 on it own is not a substratefor the BoNT/A LC because it does not contain the sequence recognizedby the active site. Accordingly no cleavage product was detected inany of our experiments.


Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity.

Xue S, Javor S, Hixon MS, Janda KD - Biochemistry (2014)

Structure of BoNT/A-LC (424 a.a. resolved, green)and SNAP-25 fragment(64 a.a. C-terminal, 59 a.a. resolved). BAP-24 region is shown inred, while the remaining SNAP-25 residues are shown in cyan and theα-exosite in blue (PDB 1XTG).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222541&req=5

fig1: Structure of BoNT/A-LC (424 a.a. resolved, green)and SNAP-25 fragment(64 a.a. C-terminal, 59 a.a. resolved). BAP-24 region is shown inred, while the remaining SNAP-25 residues are shown in cyan and theα-exosite in blue (PDB 1XTG).
Mentions: To explore how α-exosite binding influences the activityof BoNT/A LC, a probe was designed based on the published crystalstructure of BoNT/A LC complexed with SNAP-25 (Figure 1, PDB 1XTG).6 The probe consisted of the helicalportion of SNAP-25 that binds to the α-exosite, spanning 24amino acids, which we term BAP-24 (BoNT/A LC α-exosite probe24). Additionally, within BAP-24, we highlight that the C-terminalalanine was replaced by aminoisobutyric acid, a well studied effectiveinducer of α-helical structure,17 which assists BAP-24 to adopt a helical structure (Figure 2). As anticipated, BAP-24 on it own is not a substratefor the BoNT/A LC because it does not contain the sequence recognizedby the active site. Accordingly no cleavage product was detected inany of our experiments.

Bottom Line: Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity.A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors.These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo.

View Article: PubMed Central - PubMed

Affiliation: Departments of Chemistry and Immunology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

ABSTRACT
Botulinum neurotoxin serotype A (BoNT/A) is one of the most lethal toxins known. Its extreme toxicity is due to its light chain (LC), a zinc protease that cleaves SNAP-25, a synaptosome-associated protein, leading to the inhibition of neuronal activity. Studies on BoNT/A LC have revealed that two regions, termed exosites, can play an important role in BoNT catalytic activity. A clear understanding of how these exosites influence neurotoxin catalytic activity would provide a critical framework for deciphering the mechanism of SNAP-25 cleavage and the design of inhibitors. Herein, based on the crystallographic structure of BoNT/A LC complexed with its substrate, we designed an α-exosite binding probe. Experiments with this unique probe demonstrated that α-exosite binding enhanced both catalytic activity and stability of the LC. These data help delineate why α-exosite binding is needed for SNAP-25 cleavage and also provide new insights into the extended lifetime observed for BoNT/A LC in vivo.

Show MeSH
Related in: MedlinePlus