PDBsum entry 1e1h

Hydrolase

PDB id: 1e1h

Contents

Protein chains

235 a.a. *

164 a.a. *

Metals

_ZN ×2

Waters ×768

* Residue conservation analysis

PDB id:

1e1h

Name:

Hydrolase

Title:

Crystal structure of recombinant botulinum neurotoxin type a light chain, self-inhibiting zn endopeptidase.

Structure:

Botulinum neurotoxin type a light chain. Chain: a, c. Fragment: residues 10-250. Synonym: bont/a lc. Engineered: yes. Botulinum neurotoxin type a light chain. Chain: b, d. Fragment: residues 252-416. Synonym: bont/a lc.

Source:

Clostridium botulinum. Organism_taxid: 1491. Strain: kyoto-f. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_variant: blr de-3.

Biol. unit:

Tetramer (from PDB file)

Resolution:

1.80Å R-factor: 0.196 R-free: 0.237

Authors:

M.Knapp,B.Rupp

Key ref:

B.Segelke et al. (2004). Crystal structure of Clostridium botulinum neurotoxin protease in a product-bound state: Evidence for noncanonical zinc protease activity. Proc Natl Acad Sci U S A, 101, 6888-6893. PubMed id: 15107500 DOI: 10.1073/pnas.0400584101

Date:

08-May-00 Release date: 19-Jun-03

Protein chains

Q45894  (BXA2_CLOBJ) -  Botulinum neurotoxin type A2 from Clostridium botulinum (strain Kyoto / Type A2)

Seq: 1296 a.a.

Struc: 235 a.a.*

Protein chains

Q45894  (BXA2_CLOBJ) -  Botulinum neurotoxin type A2 from Clostridium botulinum (strain Kyoto / Type A2)

Seq: 1296 a.a.

Struc: 164 a.a.

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B, C, D: E.C.3.4.24.69 - bontoxilysin.
• Reaction: Limited hydrolysis of proteins of the neuroexocytosis apparatus, synaptobrevins, SNAP25 or syntaxin. No detected action on small molecule substrates.
• Cofactor: Zn(2+)

DOI no: 10.1073/pnas.0400584101

Proc Natl Acad Sci U S A 101:6888-6893 (2004)

PubMed id: 15107500

Crystal structure of Clostridium botulinum neurotoxin protease in a product-bound state: Evidence for noncanonical zinc protease activity.

B.Segelke, M.Knapp, S.Kadkhodayan, R.Balhorn, B.Rupp.

ABSTRACT

Clostridium botulinum neurotoxins (BoNTs), the most potent toxins known, disrupt neurotransmission through proteolysis of proteins involved in neuroexocytosis. The light chains of BoNTs are unique zinc proteases that have stringent substrate specificity and require exceptionally long substrates. We have determined the crystal structure of the protease domain from BoNT serotype A (BoNT/A). The structure reveals a homodimer in a product-bound state, with loop F242-V257 from each monomer deeply buried in its partner's catalytic site. The loop, which acts as a substrate, is oriented in reverse of the canonical direction for other zinc proteases. The Y249-Y250 peptide bond of the substrate loop is hydrolyzed, leaving the Y249 product carboxylate coordinated to the catalytic zinc. From the crystal structure of the BoNT/A protease, detailed models of noncanonical binding and proteolysis can be derived which we propose are also consistent with BoNT/A binding and proteolysis of natural substrate synaptosome-associated protein of 25 kDa (SNAP-25). The proposed BoNT/A substrate-binding mode and catalytic mechanism are markedly different from those previously proposed for the BoNT serotype B.

Selected figure(s)

Figure 4.
Fig. 4. The S1' specificity pocket. A ball-and-stick representation of a modeled interaction between P1' and S1' for the homodimer with Y250 in the proposed S1' pocket. Hydrogen bonds and ionic interactions are highlighted with yellow dashes. This figure was generated with MIDAS (36).

Figure 5.
Fig. 5. Catalytic mechanism. (a) A stereo view of thermolysin residues [yellow, with side chain O atoms, red, and side chain N atoms, blue; PDB 1TMN [PDB] (28)] involved in catalysis is shown superimposed on the homologous residues from BoNT/A (shown in cyan). P1 and P2 of the cleavage product from pseudosubstrate are also shown with the product carboxylate coordinated to the catalytic zinc. BoNT/A LC Y365 (magenta) is shown in the conformation from holotoxin. This figure was generated with MIDAS (36). (b) A schematic illustration of the proposed catalytic mechanism of 250s loop hydrolysis in the BoNT/A homodimer is shown. This figure was generated with ISIS DRAW.

Figures were selected by the author.