PDBsum entry 2vc3

Hydrolase

PDB id: 2vc3

Contents

Protein chain

263 a.a. *

Ligands

ACT

SO4 ×2

Waters ×467

* Residue conservation analysis

PDB id:

2vc3

Name:

Hydrolase

Title:

Ricin a-chain (recombinant) e177d mutant with a bound acetate

Structure:

Ricin a chain. Chain: a. Fragment: residues 36-302. Synonym: ricin, rrna n-glycosidase. Engineered: yes. Mutation: yes

Source:

Ricinus communis. Castor bean. Organism_taxid: 3988. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.60Å R-factor: 0.177 R-free: 0.209

Authors:

C.J.Marsden,V.Fulop

Key ref:

S.C.Allen et al. (2007). The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae. Febs J, 274, 5586-5599. PubMed id: 17916187

Date:

18-Sep-07 Release date: 16-Oct-07

Protein chain

P02879  (RICI_RICCO) -  Ricin from Ricinus communis

Seq: 576 a.a.

Struc: 263 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.2.2.22 - rRNA N-glycosylase.
• Reaction: Endohydrolysis of the N-glycosidic bond at one specific adenosine on the 28S rRNA.

Febs J 274:5586-5599 (2007)

PubMed id: 17916187

The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae.

S.C.Allen, K.A.Moore, C.J.Marsden, V.Fülöp, K.G.Moffat, J.M.Lord, G.Ladds, L.M.Roberts.

ABSTRACT

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37 degrees C, whereas the same cells did not grow at 23 degrees C. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23 degrees C but not at 37 degrees C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterization of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three-dimensional structure. The potential utility of these mutants is discussed.