Colicin-E3

 

Colicin E3 kills Escherichia coli cells by binding to a surface receptor, entering the cell and inactivating protein biosynthetic machinery by ribonucleolytic cleavage of the ribosomal subunit 16S rRNA. The mechanism shows homology to ribonuclease T1.

 

Reference Protein and Structure

Sequences
P00646 UniProt (3.1.-.-)
P02984 UniProt IPR024575 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli (Bacteria) Uniprot
PDB
1jch - Crystal Structure of Colicin E3 in Complex with its Immunity Protein (3.02 Å) PDBe PDBsum 1jch
Catalytic CATH Domains
3.10.380.10 CATHdb (see all for 1jch)
Click To Show Structure

Enzyme Reaction (EC:3.1.-.-)

RNA (poly(A))
CHEBI:8756ChEBI
+
water
CHEBI:15377ChEBI
→
RNA (poly(A))
CHEBI:8756ChEBI
+
RNA (poly(A))
CHEBI:8756ChEBI

Enzyme Mechanism

Introduction

The mechanism of colicin E3 involves a Glu-His-Asp catalytic triad. The carboxylate of Glu 517 acts as a general base catalyst to abstract a proton from the ribose O2'-hydroxyl of adenine 1493 of the ribosomal subunit. This increases the nucleophilicity of the oxygen atom so that it makes a nucleophilic attack on the phosphate to form a cyclic 2'-3' phosphate with a negative charge. The pentacoordinate cyclic phosphate intermediate is stabilised by Arg 545. Asp 510 activates His 513 to enable it to act as a general acid catalyst to facilitate loss of the leaving group by donation of a proton to the leaving 5'-OH RNA fragment upon cleavage. His 513 then acts as a general base catalyst to activate a water molecule for nucleophilic attack on the phosphorous atom of the 2'-3' cyclic phosphate. Glu 517 acts as a general acid catalyst to donate its proton to the O2' atom.

Catalytic Residues Roles

UniProt PDB* (1jch)
Asp510 Asp510A Activates His 513. electrostatic stabiliser, increase acidity
His513 His513A Functions as a general acid/base catalyst to promote scissile bond cleavage and activation of water as a nucleophile. promote heterolysis, proton acceptor, proton donor
Glu517 Glu517A Acts as a general acid/base catalyst to activate the ribose O2' oxygen to act as a nucleophile, and protonate the same oxygen again after cleavage. increase nucleophilicity, promote heterolysis, proton acceptor, proton donor
Arg545 Arg545A Stabilises the pentacoordinate cyclic phosphate intermediate. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

overall reactant used, intramolecular nucleophilic addition, proton transfer, cyclisation, overall product formed, heterolysis, bimolecular nucleophilic addition, native state of enzyme regenerated, decyclisation

References

  1. Soelaiman S et al. (2001), Mol Cell, 8, 1053-1062. Crystal structure of colicin E3: implications for cell entry and ribosome inactivation. PMID:11741540.
  2. Ng CL et al. (2010), Nat Struct Mol Biol, 17, 1241-1246. Structural basis for 16S ribosomal RNA cleavage by the cytotoxic domain of colicin E3. DOI:10.1038/nsmb.1896. PMID:20852642.
  3. Zarivach R et al. (2002), Biochimie, 84, 447-454. On the interaction of colicin E3 with the ribosome. DOI:10.1016/s0300-9084(02)01449-9. PMID:12423788.

Step 1. Glu517 acts as a base abstracting a proton from the 2' hydroxyl group of the A1493 sub-unit. This increases the nucleophilicity of the group allowing it to attack the phosphate group and form a penta-covalent intermediate which is stabilized by Arg545.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg545A electrostatic stabiliser
Glu517A increase nucleophilicity
Glu517A proton acceptor

Chemical Components

overall reactant used, ingold: intramolecular nucleophilic addition, proton transfer, cyclisation

Step 2. Asp510 activates His513 enabling His513 to act as an acid donating a proton to the 5' hydroxyl. The penta-covalent intermediate collapses and the bond between the phosphate and the 5' hydroxyl is cleaved.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp510A electrostatic stabiliser, increase acidity
His513A promote heterolysis
Arg545A electrostatic stabiliser
His513A proton donor

Chemical Components

overall product formed, heterolysis, proton transfer

Step 3. His513 now acts as a base activating a water molecule for nucleophillic attack on the phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp510A electrostatic stabiliser
Arg545A electrostatic stabiliser
His513A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 4. The penta-covalent intermediate collapses breaking the bond between the 2' hydroxyl and the phosphate with Glu517 acting as an acid.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg545A electrostatic stabiliser
Glu517A promote heterolysis, proton donor

Chemical Components

heterolysis, overall product formed, proton transfer, native state of enzyme regenerated, decyclisation
Download: Image, Marvin File

Step 1. Glu517 acts as a base abstracting a proton from the 2' hydroxyl group of the A1493 sub-unit. This increases the nucleophilicity of the group allowing it to attack the phosphate group and form a penta-covalent intermediate which is stabilized by Arg545.

Step 2. Asp510 activates His513 enabling His513 to act as an acid donating a proton to the 5' hydroxyl. The penta-covalent intermediate collapses and the bond between the phosphate and the 5' hydroxyl is cleaved.

Step 3. His513 now acts as a base activating a water molecule for nucleophillic attack on the phosphate.

Step 4. The penta-covalent intermediate collapses breaking the bond between the 2' hydroxyl and the phosphate with Glu517 acting as an acid.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, James Willey