Abstract
Ricin A chain (RTA) depurinates the sarcin/ricin loop (SRL) by interacting with the C-termini of the ribosomal P stalk. The ribosome interaction site and the active site are located on opposite faces of RTA. The interaction with P proteins allows RTA to depurinate the SRL on the ribosome at physiological pH with an extremely high activity by orienting the active site towards the SRL. Therefore, if an inhibitor disrupts RTA–ribosome interaction by binding to the ribosome binding site of RTA, it should inhibit the depurination activity. To test this model, we synthesized peptides mimicking the last 3 to 11 amino acids of P proteins and examined their interaction with wild-type RTA and ribosome binding mutants by Biacore. We measured the inhibitory activity of these peptides on RTA-mediated depurination of yeast and rat liver ribosomes. We found that the peptides interacted with the ribosome binding site of RTA and inhibited depurination activity by disrupting RTA–ribosome interactions. The shortest peptide that could interact with RTA and inhibit its activity was four amino acids in length. RTA activity was inhibited by disrupting its interaction with the P stalk without targeting the active site, establishing the ribosome binding site as a new target for inhibitor discovery.
Highlights
Ricin (E.C. 3.2.2.22), produced by the castor bean (Ricinus communis), belongs to a group of toxic proteins called ribosome-inactivating proteins (RIPs) that include major human pathogens, such as Escherichia coli and Shigella producing Shiga toxins (Stxs)
We showed that the ribosome binding site and the active site are located on opposite faces of RTA and based on these results we proposed a molecular model for depurination of the sarcin/ricin loop (SRL) by RTA [22]
We previously showed that arginines at the RTA/RTB interface contribute to fast electrostatic interactions with the C-terminal domain (CTD) of the P proteins, indicating that the negatively charged motif plays an important role in the interaction of RTA with the ribosome [29]
Summary
Ricin (E.C. 3.2.2.22), produced by the castor bean (Ricinus communis), belongs to a group of toxic proteins called ribosome-inactivating proteins (RIPs) that include major human pathogens, such as Escherichia coli and Shigella producing Shiga toxins (Stxs). The interaction of RTA with P proteins is critical for ribosome binding, depurination of the SRL, and toxicity of RTA in the yeast Saccharomyces cerevisiae and in human cells [5,21]. A 17-mer peptide mimicking the CTD of the human ribosomal stalk P2 protein was shown to inhibit the activity of the A1 subunit of Shiga toxin 1 (Stx1A1) in an in vitro translation assay [6]. We previously showed that arginines at the RTA/RTB interface contribute to fast electrostatic interactions with the CTD of the P proteins, indicating that the negatively charged motif plays an important role in the interaction of RTA with the ribosome [29]. Toxins 2018, 10, x FOR PEER REVIEW of peptides corresponding to the last 3 to 11 amino acids of human P proteins with RTA and examined their ability to inhibit the depurination activity of RTA.
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