3MK9

Stabilized Ricin Immunogen 1-33/44-198

Summary for 3MK9

• Entry DOI: 10.2210/pdb3mk9/pdb
• Related: 3LC9
• Descriptor: Ricin, SULFATE ION (3 entities in total)
• Functional Keywords: disulfide bond, hydrolase, nucleotide-binding, plant defense, protein synthesis inhibitor, toxin, immunogen
• Biological source: Ricinus communis (Castor bean)
• Total number of polymer chains: 1
• Total formula weight: 21541.30

Authors

Legler, P.M.,Compton, J.R.,Millard, C.B. (deposition date: 2010-04-14, release date: 2010-11-10, Last modification date: 2024-10-09)

Primary citation

Compton, J.R.,Legler, P.M.,Clingan, B.V.,Olson, M.A.,Millard, C.B.
Introduction of a disulfide bond leads to stabilization and crystallization of a ricin immunogen.
Proteins, 79:1048-1060, 2011

PubMed Abstract: 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°C compared with RTA1-33/44-198 and by ~13°C compared with RTA. Prolonged stability studies of the R48C/T77C variant (> 60 days at 37°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.

PubMed: 21387408
DOI: 10.1002/prot.22933

Experimental method

X-RAY DIFFRACTION (2.08 Å)