Article

Catalytic features of the botulinum neurotoxin A light chain revealed by high resolution structure of an inhibitory peptide complex.

Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Biochemistry (impact factor: 3.42). 06/2008; 47(21):5736-45. DOI:10.1021/bi8001067 pp.5736-45
Source: PubMed

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 A-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 gamma 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 A 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.

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    PLoS ONE 01/2009; 4(11):e7730. · 4.09 Impact Factor
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Keywords

A-resolution X-ray crystal structure
 
catalytic water molecule
 
cleaving SNAP-25
 
Clostridium botulinum neurotoxin serotype
 
Cys S gamma atom
 
cysteine sulfur
 
enzyme-peptide structures available
 
first structure
 
inhibitory peptide
 
metal ion
 
neuromuscular junction
 
peptide-bound structure
 
peptides bind
 
protease reaction
 
second water molecule
 
SNARE proteins
 
sulfenic acid form
 
unstable nature
 
Zn(II)-bound uncomplexed enzyme
 
Zn(II)-dependent metalloprotease
 

Nicholas R Silvaggi