7TH2

Single-domain VHH intrabodies neutralize ricin toxin

Summary for 7TH2

• Entry DOI: 10.2210/pdb7th2/pdb
• Descriptor: Ricin A chain, VHH antibody, CHLORIDE ION, ... (7 entities in total)
• Functional Keywords: antibody-antigen complex, immune system, immune system-toxin complex, immune system/toxin
• Biological source: Ricinus communis (castor bean); More
• Total number of polymer chains: 4
• Total formula weight: 90358.89

Authors

Rudolph, M.J.,Mantis, N. (deposition date: 2022-01-10, release date: 2022-10-12, Last modification date: 2024-10-23)

Primary citation

Czajka, T.F.,Vance, D.J.,Davis, S.,Rudolph, M.J.,Mantis, N.J.
Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins.
J.Biol.Chem., 298:101742-101742, 2022

PubMed Abstract: 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. 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 vitro assays confirmed that these VHs block RTA-P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as "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 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.

PubMed: 35182523
DOI: 10.1016/j.jbc.2022.101742

Experimental method

X-RAY DIFFRACTION (1.838 Å)