Catalytic features of the botulinum neurotoxin A light chain revealed by high resolution structure of an inhibitory peptide complex
- PMID:18457419
- DOI: 10.1021/bi8001067
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 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.
Similar articles
- Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate.Fu Z, Chen S, Baldwin MR, Boldt GE, Crawford A, Janda KD, Barbieri JT, Kim JJ.Fu Z, et al.Biochemistry. 2006 Jul 25;45(29):8903-11. doi: 10.1021/bi060786z.Biochemistry. 2006.PMID:16846233
- Quinolinol and peptide inhibitors of zinc protease in botulinum neurotoxin A: effects of zinc ion and peptides on inhibition.Lai H, Feng M, Roxas-Duncan V, Dakshanamurthy S, Smith LA, Yang DC.Lai H, et al.Arch Biochem Biophys. 2009 Nov;491(1-2):75-84. doi: 10.1016/j.abb.2009.09.008. Epub 2009 Sep 20.Arch Biochem Biophys. 2009.PMID:19772855
- Substrate recognition strategy for botulinum neurotoxin serotype A.Breidenbach MA, Brunger AT.Breidenbach MA, et al.Nature. 2004 Dec 16;432(7019):925-9. doi: 10.1038/nature03123. Epub 2004 Dec 12.Nature. 2004.PMID:15592454
- New insights into clostridial neurotoxin-SNARE interactions.Breidenbach MA, Brunger AT.Breidenbach MA, et al.Trends Mol Med. 2005 Aug;11(8):377-81. doi: 10.1016/j.molmed.2005.06.012.Trends Mol Med. 2005.PMID:16006188Review.
- D-Glyceraldehyde-3-phosphate dehydrogenase: structural basis and functional role of the acyl transfer reactions.Nagradova NK, Schmalhausen EV.Nagradova NK, et al.Biochemistry (Mosc). 1998 May;63(5):504-15.Biochemistry (Mosc). 1998.PMID:9632884Review.
Cited by
- Identification of 3-hydroxy-1,2-dimethylpyridine-4(1H)-thione as a metal-binding motif for the inhibition of botulinum neurotoxin A.Lin L, Turner LD, Šilhár P, Pellett S, Johnson EA, Janda KD.Lin L, et al.RSC Med Chem. 2020 Nov 12;12(1):137-143. doi: 10.1039/d0md00320d. eCollection 2021 Jan 1.RSC Med Chem. 2020.PMID:34046606Free PMC article.
- Clostridial neurotoxins: mechanism of SNARE cleavage and outlook on potential substrate specificity reengineering.Binz T, Sikorra S, Mahrhold S.Binz T, et al.Toxins (Basel). 2010 Apr;2(4):665-82. doi: 10.3390/toxins2040665. Epub 2010 Apr 13.Toxins (Basel). 2010.PMID:22069605Free PMC article.Review.
- Catch and Anchor Approach To Combat Both Toxicity and Longevity of Botulinum Toxin A.Lin L, Olson ME, Sugane T, Turner LD, Tararina MA, Nielsen AL, Kurbanov EK, Pellett S, Johnson EA, Cohen SM, Allen KN, Janda KD.Lin L, et al.J Med Chem. 2020 Oct 8;63(19):11100-11120. doi: 10.1021/acs.jmedchem.0c01006. Epub 2020 Sep 18.J Med Chem. 2020.PMID:32886509Free PMC article.
- Potent new small-molecule inhibitor of botulinum neurotoxin serotype A endopeptidase developed by synthesis-based computer-aided molecular design.Pang YP, Vummenthala A, Mishra RK, Park JG, Wang S, Davis J, Millard CB, Schmidt JJ.Pang YP, et al.PLoS One. 2009 Nov 10;4(11):e7730. doi: 10.1371/journal.pone.0007730.PLoS One. 2009.PMID:19901994Free PMC article.
- Basic tetrapeptides as potent intracellular inhibitors of type A botulinum neurotoxin protease activity.Hale M, Oyler G, Swaminathan S, Ahmed SA.Hale M, et al.J Biol Chem. 2011 Jan 21;286(3):1802-11. doi: 10.1074/jbc.M110.146464. Epub 2010 Oct 20.J Biol Chem. 2011.PMID:20961849Free PMC article.
Publication types
MeSH terms
Substances
Associated data
- Actions
- Actions
- Actions
Related information
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical