7kc9
Ricin bound to VHH antibody V5G1
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 PubMedRicin toxin kills mammalian cells with notorious efficiency. The toxin's B subunit (RTB) is a Gal/GalNAc-specific lectin that attaches to cell surfaces and promotes retrograde transport of ricin's A subunit (RTA) to the trans Golgi network (TGN) and endoplasmic reticulum (ER). RTA is liberated from RTB in the ER and translocated into the cell cytoplasm, where it functions as a ribosome-inactivating protein. While antibodies against ricin's individual subunits have been reported, we now describe seven alpaca-derived, single-domain antibodies (V(H)Hs) that span the RTA-RTB interface, including four Tier 1 V(H)Hs with IC(50) values <1 nM. Crystal structures of each V(H)H bound to native ricin holotoxin revealed three different binding modes, based on contact with RTA's F-G loop (mode 1), RTB's subdomain 2gamma (mode 2) or both (mode 3). V(H)Hs in modes 2 and 3 were highly effective at blocking ricin attachment to HeLa cells and immobilized asialofetuin, due to framework residues (FR3) that occupied the 2gamma Gal/GalNAc-binding pocket and mimic ligand. The four Tier 1 V(H)Hs also interfered with intracellular functions of RTB, as they neutralized ricin in a post-attachment cytotoxicity assay (e.g., the toxin was bound to cell surfaces before antibody addition) and reduced the efficiency of toxin transport to the TGN. We conclude that the RTA-RTB interface is a target of potent toxin-neutralizing antibodies that interfere with both extracellular and intracellular events in ricin's cytotoxic pathway. Structural Analysis of Toxin-Neutralizing, Single-Domain Antibodies that Bridge Ricin's A-B Subunit Interface.,Rudolph MJ, Poon AY, Kavaliauskiene S, Myrann AG, Reynolds-Peterson C, Davis SA, Sandvig K, Vance DJ, Mantis NJ J Mol Biol. 2021 Jul 23;433(15):167086. doi: 10.1016/j.jmb.2021.167086. Epub 2021 , Jun 3. PMID:34089718[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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