Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2

Abstract

Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome.

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.…”

“…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.…”

“…However, the two groups drew different conclusions. 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 ].…”

Pretreatment with Retro‐2 protects cells from death caused by ricin toxin by retaining the capacity of protein synthesis

“…The active form of RTA can selectively remove a single adenine from 28S rRNA. This results in further binding of elongation factor‐2 and blocking of protein synthesis, thereby causing cell death (Endo & Tsurugi, 1988; Shi et al, 2016).…”