2VC4

Ricin A-Chain (Recombinant) E177D Mutant

Summary for 2VC4

• Entry DOI: 10.2210/pdb2vc4/pdb
• Related: 1APG 1BR5 1BR6 1FMP 1IFS 1IFT 1IFU 1IL3 1IL4 1IL5 1IL9 1J1M 1OBS 1OBT 1RTC 1UQ4 1UQ5 1ZAM 1ZB0 1ZB2 2AAI 2VC3
• Descriptor: RICIN A CHAIN, GLYCEROL, SULFATE ION, ... (4 entities in total)
• Functional Keywords: glycoprotein, plant defense, protein synthesis inhibitor, toxin, lectin, hydrolase, glycosidase
• Biological source: RICINUS COMMUNIS (CASTOR BEAN)
• Total number of polymer chains: 1
• Total formula weight: 30206.95

Authors

Marsden, C.J.,Fulop, V. (deposition date: 2007-09-18, release date: 2007-10-16, Last modification date: 2023-12-13)

Primary citation

Allen, S.C.H.,Moore, K.A.H.,Marsden, C.J.,Fulop, V.,Moffat, K.G.,Lord, J.M.,Ladds, G.,Roberts, L.M.
The Isolation and Characterisation of Temperature-Dependent Ricin a Chain Molecules in Saccharomyces Cerevisiae
FEBS J., 274:5586-, 2007

PubMed 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.

PubMed: 17916187
DOI: 10.1111/J.1742-4658.2007.06080.X

Experimental method

X-RAY DIFFRACTION (1.39 Å)