Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved ␣-sarcin loop of large rRNAs. They have received attention in biological and biomedical research because of their unique biological activities toward animals and human cells as cell-killing agents. A better understanding of the depurination mechanism of RIPs could allow us to develop potent neutralizing antibodies and to design efficient immunotoxins for clinical use. Among these RIPs, ricin exhibited remarkable efficacy in depurination activity and highly conserved tertiary structure with other RIPs. It can be considered as a prototype to investigate the depurination mechanism of RIPs. In the present study, we successfully identified a novel functional domain responsible for controlling the depurination activity of ricin, which is located far from the enzymatic active site reported previously. Our study indicated that ricin A-chain mAbs binding to this domain (an ␣-helix comprising the residues 99 -106) exhibited an unusual potent neutralizing ability against ricin in vivo. To further investigate the potential role of the ␣-helix in regulating the catalytic activity of ricin, ricin A-chain variants with different flexibility of the ␣-helix were rationally designed. Our data clearly demonstrated that the flexibility of the ␣-helix is responsible for controlling the depurination activity of ricin and determining the extent of protein synthesis inhibition, suggesting that the conserved ␣-helix might be considered as a potential target for the prevention and treatment of RIP poisoning. Ribosome-inactivating proteins (RIPs)3 are depurinating rRNA N-glycosidases (E.C. 3.2.2.22) that cleave a single bond between a specific adenine and ribose of rRNA in eukaryotes (1). They are generally divided into two classes (1-3). Type I RIPs such as trichosanthin and gelonin are monomeric enzymes of ϳ30 kDa. Type II RIPs are heterodimeric proteins with an approximate molecular mass of 60 kDa, in which one polypeptide with RIP activity (A-chain) is linked by a disulfide bridge to a galactose-binding lectin (B-chain). The B-chain is able to bind to a galactose-containing receptor on the surface of sensitive cells and mediate transport of the A-chain through the secretory pathways into the cytoplasm.Ricin (a type II RIP), which has emerged as a powerful catalyst for mammalian ribosomes, is a good prototype to investigate the N-glycosidase mechanism of RIPs. Ricin A-chain (RTA) is the catalytic subunit of ricin, which catalyzes the depurination of an invariant adenosine residue, A 4324 , within the GA 4324 GA tetraloop motif of the highly conserved sarcinricin loop of eukaryotic 28 S rRNA (4). Most previous studies have focused on the role of the active-site residues that are crucial for catalytic activity of RIPs. Day et al. (5) have presented the crystal structure of ricin, indicating that RTA has a prominent cleft able to recognize the target rRNA stem-loop. Sitedirected mutagenesis, as well as analysis by systematic deletion ...

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