3C8B: Crystal Structure Of The Catalytic Domain Of Botulinum Neurotoxin Serotype A With Inhibitory Peptide Rrgi

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6-1.8 A. These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr(366) and NH(2) of Arg(363) are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe(194), Thr(215), Thr(220), Asp(370), and Arg(363). The K(i) of the best inhibitory tetrapeptide is 157 nm.
PDB ID: 3C8BDownload
MMDB ID: 87063
PDB Deposition Date: 2008/2/11
Updated in MMDB: 2010/12
Experimental Method:
x-ray diffraction
Resolution: 1.47  Å
Source Organism:
synthetic construct
Similar Structures:
Biological Unit for 3C8B: dimeric; determined by author and by software (PISA)
Molecular Components in 3C8B
Label Count Molecule
Proteins (2 molecules)
Botulinum Neurotoxin a Light Chain(Gene symbol: atx)
Molecule annotation
Inhibitor Peptide Rrgi
Molecule annotation
Chemicals (4 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB