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Keywords:

  • aggregation;
  • unfolding;
  • subunit vaccine;
  • toxin;
  • protein engineering;
  • crystal packing

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 2.3Å or 2.1 Å 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. Proteins 2011. Published 2010 Wiley-Liss, Inc.