Structural highlights
Function
RICI_RICCO Ricin is highly toxic to animal cells and to a lesser extent to plant cells. The A chain acts as a glycosidase that removes a specific adenine residue from an exposed loop of the 28S rRNA (A4324 in mammals), leading to rRNA breakage. As this loop is involved in elongation factor binding, modified ribosomes are catalytically inactive and unable to support protein synthesis. The A chain can inactivate a few thousand ribosomes per minute, faster than the cell can make new ones. Therefore a single A chain molecule can kill an animal cell. The B chain binds to beta-D-galactopyranoside moieties on cell surface glycoproteins and glycolipids and facilitates the entry into the cell of the A chain; B chains are also responsible for cell agglutination (Lectin activity).
Publication Abstract from PubMed
RTA1-33/44-198 is a catalytically inactive, single-domain derivative of the ricin toxin A-chain (RTA) engineered to serve as a stable protein scaffold for presentation of native immunogenic epitopes (Olson et al., Protein Eng Des Sel 2004;17:391-397). To improve the stability and solubility of RTA1-33/44-198 further, we have undertaken the design challenge of introducing a disulfide (SS) bond. Nine pairs of residues were selected for placement of the SS-bond based on molecular dynamics simulation studies of the modeled single-domain chain. Disulfide formation at either of two positions (R48C/T77C or V49C/E99C) involving a specific surface loop (44-55) increased the protein melting temperature by ~5 degrees C compared with RTA1-33/44-198 and by ~13 degrees C compared with RTA. Prolonged stability studies of the R48C/T77C variant (> 60 days at 37 degrees C, pH 7.4) confirmed a > 40% reduction in self-aggregation compared with RTA1-33/44-198 lacking the SS-bond. The R48C/T77C variant retained affinity for anti-RTA antibodies capable of neutralizing ricin toxin, including a monoclonal that recognizes a human B-cell epitope. Introduction of either R48C/T77C or V49C/E99C promoted crystallization of RTA1-33/44-198, and the X-ray structures of the variants were solved to 2.3 A or 2.1 A resolution, respectively. The structures confirm formation of an intramolecular SS-bond, and reveal a single-domain fold that is significantly reduced in volume compared with RTA. Loop 44 to 55 is partly disordered as predicted by simulations, and is positioned to form self-self interactions between symmetry-related molecules. We discuss the importance of RTA loop 34 to 55 as a nucleus for unfolding and aggregation, and draw conclusions for ongoing structure-based minimalist design of RTA-based immunogens.
Introduction of a disulfide bond leads to stabilization and crystallization of a ricin immunogen.,Compton JR, Legler PM, Clingan BV, Olson MA, Millard CB Proteins. 2011 Apr;79(4):1048-60. doi: 10.1002/prot.22933. Epub 2011 Jan, 5. PMID:21387408[1]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ Compton JR, Legler PM, Clingan BV, Olson MA, Millard CB. Introduction of a disulfide bond leads to stabilization and crystallization of a ricin immunogen. Proteins. 2011 Apr;79(4):1048-60. doi: 10.1002/prot.22933. Epub 2011 Jan, 5. PMID:21387408 doi:10.1002/prot.22933
