3mk9 | pdb_00003mk9

Stabilized Ricin Immunogen 1-33/44-198

spinqualitylabelsall models 3mk9, resolution 2.08Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3mk9 is a 1 chain structure with sequence from Ricinus communis. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: SO4 Related: 3lc9 Activity: rRNA N-glycosylase, with EC number 3.2.2.22 Resources: FirstGlance, OCA, RCSB, PDBsum 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Ricin

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