PDBsum entry 3c8b

Hydrolase/hydrolase inhibitor

PDB id: 3c8b

Contents

Protein chain

423 a.a. *

Ligands

ARG-ARG-GLY-ILE-NH2

SO4 ×3

Metals

_ZN

Waters ×372

* Residue conservation analysis

PDB id:

3c8b

Name:

Hydrolase/hydrolase inhibitor

Title:

Crystal structure of the catalytic domain of botulinum neurotoxin serotype a with inhibitory peptide rrgi

Structure:

Botulinum neurotoxin a light chain. Chain: a. Fragment: residues 1-424. Synonym: bontoxilysin-a. Engineered: yes. Inhibitor peptide rrgi. Chain: b. Engineered: yes

Source:

Clostridium botulinum. Organism_taxid: 1491. Strain: hall. Atcc: 3502. Gene: bota. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Synthetic construct.

Resolution:

1.47Å R-factor: 0.204 R-free: 0.216

Authors:

D.Kumaran,S.Swaminathan

Key ref:

D.Kumaran et al. (2008). Structure- and substrate-based inhibitor design for Clostridium botulinum neurotoxin serotype A. J Biol Chem, 283, 18883-18891. PubMed id: 18434312 DOI: 10.1074/jbc.M801240200

Date:

11-Feb-08 Release date: 22-Apr-08

Protein chain

P0DPI1  (BXA1_CLOBH) -  Botulinum neurotoxin type A from Clostridium botulinum (strain Hall / ATCC 3502 / NCTC 13319 / Type A)

Seq: 1296 a.a.

Struc: 423 a.a.

Enzyme reactions

• Enzyme class: 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: 10.1074/jbc.M801240200

J Biol Chem 283:18883-18891 (2008)

PubMed id: 18434312

Structure- and substrate-based inhibitor design for Clostridium botulinum neurotoxin serotype A.

D.Kumaran, R.Rawat, M.L.Ludivico, S.A.Ahmed, S.Swaminathan.

ABSTRACT

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.

Selected figure(s)

Figure 4.
FIGURE 4. Schematic diagram of molecular interactions between Balc424 and tetrapeptide inhibitors. The interactions between Balc424 active site residues and inhibitor peptides are shown. A, RRGC; B, RRGM; C, RRGL; and D, RRGI. Black, red, blue, yellow, and green colored circles represent carbon, oxygen, nitrogen, sulfur, and sodium atoms, respectively. For clarity, zinc coordination and water molecules involved in the interactions at the active site are not shown. The figure was prepared with Ligplot (47).

Figure 7.
FIGURE 7. Electrostatic potential surface representation of active site cavity. Models of RRGC (green), RRGM (light green), RRGL (coral), and RRGI (magenta) with sulfate ions are superposed. Red and blue indicate negative ( -0.5 V) and positive ( +0.5 V) electrostatic potential. The figure was prepared with CCP4MG (50). The orientation is similar to Fig. 3A.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 18883-18891) copyright 2008.

Figures were selected by an automated process.