Introduction of a disulfide bond leads to stabilization and crystallization of a ricin immunogen

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.