Structural insights into the interaction of the ribosomal P stalk protein P2 with a type II ribosome-inactivating protein ricin

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.

Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding

“…The C-terminal ends of the ribosomal stalk P proteins interact with a small well-defined hydrophobic pocket on the face of RTA opposite to the active site [ 27 , 28 ]. We show here that peptides derived from the conserved CTD of P proteins can disrupt RTA–ribosome interactions.…”

“…The longest peptide tested was P11 because this is the smallest peptide reported to inhibit the activity of Stx1 [ 6 ]. Since the C-terminal end of this peptide is critical for RTA interaction [ 27 , 28 ], we deleted one amino acid at a time starting from the N-terminal end. We found that the longer peptides had higher affinity and inhibitory activity compared to the shorter peptides.…”

“…Although the amino acid sequences of the A1 subunit of Stx1 and Stx2 are only 21% and 20% identical to RTA, respectively, they are structurally and functionally very similar to RTA [ 26 ]. Recently, the structure of RTA together with the last six amino acids of P proteins was resolved by two different groups using different strategies (PDB IDs: 5GU4 and 5DDZ) [ 27 , 28 ]. Although 9-mer, 11-mer [ 27 ], or 10-mer [ 28 ] P stalk peptides were used in the studies, the structures resolved by both groups showed that only the last six residues interact with RTA.…”

“…Recently, the structure of RTA together with the last six amino acids of P proteins was resolved by two different groups using different strategies (PDB IDs: 5GU4 and 5DDZ) [ 27 , 28 ]. Although 9-mer, 11-mer [ 27 ], or 10-mer [ 28 ] P stalk peptides were used in the studies, the structures resolved by both groups showed that only the last six residues interact with RTA. The last six residues (G 110 FGLFD 115 ) bind to a hydrophobic pocket on RTA in a unique conformation.…”

“…One group postulated that this motif contributes to the interaction because GST-tagged peptides, which contained the D 106 D 107 D 108 motif, showed higher affinities for RTA than those without this motif [ 27 ]. In contrast, the other group concluded that the D 106 D 107 D 108 motif does not contribute to the RTA interaction because they did not observe any difference in the pull-down experiments with His-tagged RTA when this motif was mutated or deleted [ 28 ]. 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 ].…”