7th3
Single-domain VHH intrabodies neutralize ricin toxin
Structural highlights
FunctionRICI_RICCO Ricin is highly toxic to animal cells and to a lesser extent to plant cells. The A chain acts as a glycosidase that removes a specific adenine residue from an exposed loop of the 28S rRNA (A4324 in mammals), leading to rRNA breakage. As this loop is involved in elongation factor binding, modified ribosomes are catalytically inactive and unable to support protein synthesis. The A chain can inactivate a few thousand ribosomes per minute, faster than the cell can make new ones. Therefore a single A chain molecule can kill an animal cell. The B chain binds to beta-D-galactopyranoside moieties on cell surface glycoproteins and glycolipids and facilitates the entry into the cell of the A chain; B chains are also responsible for cell agglutination (Lectin activity). Publication Abstract from PubMedDuring 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. 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 (V(H)Hs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such V(H)Hs (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 vitro assays confirmed that these V(H)Hs block RTA-P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as "intrabodies," these V(H)Hs rendered cells resistant to ricin intoxication. One V(H)H (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 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. Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins.,Czajka TF, Vance DJ, Davis S, Rudolph MJ, Mantis NJ J Biol Chem. 2022 Apr;298(4):101742. doi: 10.1016/j.jbc.2022.101742. Epub 2022 , Feb 17. PMID:35182523[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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