During ricin intoxication in mammalian cells, ricin’s enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin-ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein (RIP). It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P stalk proteins, whose C-terminal domains (CTD) interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here we characterized a collection of single-domain antibodies (VHs) whose epitopes overlap with the P stalk binding pocket on RTA. The crystal structures of three such VHs (V9E1, V9F9, and V9B2) in complex with RTA not only revealed occlusion of the ribosomal P stalk binding pocket, but also structural mimicry of CTD peptides by complementarity-determining region (CDR) 3. In vitro assays confirmed that these VHs block RTA-P stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as cytoplasmic “intrabodies,” these VHs rendered cells resistant to ricin intoxication. One VH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P stalk binding pocket, was not effective at neutralizing ricin, nor did it protect ribosomes from RTA’s RIP activity in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P stalk proteins as a requisite event in ricin-induced ribosome inactivation and subsequent cell death.
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