skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex

Abstract

The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 Angstroms-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S? atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-boundmore » structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 Angstroms resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
959812
Report Number(s):
BNL-82798-2009-JA
Journal ID: ISSN 0006-2960; BICHAW; TRN: US201016%%956
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 47; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ACETYLCHOLINE; ATOMS; CHAINS; CLEAVAGE; CLOSTRIDIUM BOTULINUM; CRYSTAL STRUCTURE; CYSTEINE; DESIGN; ENZYMES; OXIDATION; OXYGEN; PEPTIDES; PROTEINS; RESOLUTION; SUBSTRATES; SULFUR; TOXINS; WATER; national synchrotron light source

Citation Formats

Silvaggi, N, Wilson, D, Tzipori, S, and Allen, K. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex. United States: N. p., 2008. Web. doi:10.1021/bi8001067.
Silvaggi, N, Wilson, D, Tzipori, S, & Allen, K. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex. United States. doi:10.1021/bi8001067.
Silvaggi, N, Wilson, D, Tzipori, S, and Allen, K. Tue . "Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex". United States. doi:10.1021/bi8001067.
@article{osti_959812,
title = {Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex},
author = {Silvaggi, N and Wilson, D and Tzipori, S and Allen, K},
abstractNote = {The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 Angstroms-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S? atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 Angstroms resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.},
doi = {10.1021/bi8001067},
journal = {Biochemistry},
issn = {0006-2960},
number = ,
volume = 47,
place = {United States},
year = {2008},
month = {1}
}