1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit.

Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA's depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.

Generation, characterization, and epitope mapping of neutralizing and protective monoclonal antibodies against staphylococcal enterotoxin B-induced lethal shock.

T-cell stimulating activity of Staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases including SEB mediated shock. SEB is one of the most potent superantigens known and treatment of SEB induced shock remains a challenge. We generated and characterized murine monoclonal antibodies (mAbs) to SEB in mice. We tested mAbs neutralize mitogenic effects of SEB in vitro and in vivo with T-cell proliferation assays and 2 murine models for SEB induced lethal shock (SEBILS). Epitope mapping suggests that all these mAbs recognize conformational epitopes that are destroyed by deleting the C terminus of the protein. Further site-directed mutagenesis identified potential residues involved in binding to SEB that differ between Methicillin resistant and sensitive Staphylococcus aureus strains. Only mAb 20B1 was effective as a monotherapy in treating SEBILS in HLA DR3 transgenic mice, which exhibit enhanced sensitivity to SEB. It is noteworthy that mAbs, 14G8 and 6D3 were not protective when given alone in the HLA DR3 mice but their efficacy of protection could be greatly enhanced when mAbs were co-administered simultaneously. Our data suggest combinations of defined mAbs may constitute a better treatment strategy and provide a new insight for the development of passive immunotherapy.

NMR characterization of a peptide model provides evidence for significant structure in the unfolded state of the villin headpiece helical subdomain.

The villin headpiece subdomain (HP36) is the smallest naturally occurring protein that folds cooperatively. The protein folds on a microsecond time scale. Its small size and very rapid folding have made it a popular target for biophysical studies of protein folding. Temperature-dependent one-dimensional (1D) NMR studies of the full-length protein together with CD and 1D NMR studies of the 21-residue peptide fragment (HP21) derived from HP36 have shown that there is significant structure in the unfolded state of HP36 and have demonstrated that HP21 is a good model of these interactions. Here, we characterized the model peptide HP21 in detail by two-dimensional NMR. Strongly upfield shifted C(alpha) protons, the magnitude of the 3J(NH,alpha) coupling constants, and the pattern of backbone-backbone and backbone-side chain NOEs indicate that the ensemble of structures populated by HP21 contains alpha-helical structure and native as well as non-native hydrophobic contacts. The hydrogen-bonded secondary structure inferred from the NOEs is, however, not sufficient to confer significant protection against amide H-D exchange. These studies indicate that there is significant secondary structure and hydrophobic clustering in the unfolded state of HP36. The implications for the folding of HP36 are discussed.