Abstract
Ricin is a type II ribosome-inactivating protein (RIP) that depurinates A4324 at the sarcin-ricin loop of 28 S ribosomal RNA (rRNA), thus inactivating the ribosome by preventing elongation factors from binding to the GTPase activation centre. Recent studies have disclosed that the conserved C-terminal domain (CTD) of eukaryotic ribosomal P stalk proteins is involved in the process that RIPs target ribosome. However, the details of the molecular interaction between ricin and P stalk proteins remain unknown. Here, we report the structure of ricin-A chain (RTA) in a complex with the CTD of the human ribosomal protein P2. The structure shows that the Phe111, Leu113 and Phe114 residues of P2 insert into a hydrophobic pocket formed by the Tyr183, Arg235, Phe240 and Ile251 residues of RTA, while Asp115 of P2 forms hydrogen bonds with Arg235 of RTA. The key residues in RTA and P2 for complex formation were mutated, and their importance was determined by pull-down assays. The results from cell-free translation assays further confirmed that the interaction with P stalk proteins is essential for the inhibition of protein synthesis by RTA. Taken together, our results provide a structural basis that will improve our understanding of the process by which ricin targets the ribosome, which will benefit the development of effective small-molecule inhibitors for use as therapeutic agents.
Highlights
In humans, the ribosomal P stalk consists of P0, P1 and P2 proteins that form a pentameric complex i.e., P0(P1)2(P2)[210]
We demonstrate that RTA interacts with the human ribosomal P stalk protein P2 with a binding affinity of 4.51 μM and mainly recognises the highly conserved C-terminal tail of P2
Our results suggest that the conserved DDDM motif of P2 is not involved in the interaction with RTA, but that two consecutive residues, Leu and Phe, in the conserved C-terminal tail of ribosomal P stalk proteins are critical for the interaction with RTA
Summary
The ribosomal P stalk consists of P0, P1 and P2 proteins that form a pentameric complex i.e., P0(P1)2(P2)[210]. A distinctive feature of P stalk proteins is that they contain a highly conserved set of 11 residues (SDDDMGFGLFD) at the C-terminal tail of P2 that recruits elongation factors and interacts with several RIPs, such as trichosanthin and Shiga toxin 114,15. We demonstrate that RTA interacts with the human ribosomal P stalk protein P2 with a binding affinity of 4.51 μM and mainly recognises the highly conserved C-terminal tail of P2 (residues 106–115). Our results suggest that the conserved DDDM motif of P2 is not involved in the interaction with RTA, but that two consecutive residues, Leu and Phe, in the conserved C-terminal tail of ribosomal P stalk proteins are critical for the interaction with RTA. Small-molecule inhibitors that mimic the 3D conformation of these two consecutive residues deserve further consideration for clinical use in the therapy of ricin poisoning
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