Abstract:

<p>During ricin intoxication in mammalian cells, ricin&#39;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. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains 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 revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In&nbsp;vitro assays confirmed that these VHs block RTA-P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as &quot;intrabodies,&quot; 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 unable to neutralize ricin within cells or protect ribosomes from RTA in&nbsp;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.</p>