6cwg
Ricin catalytic subunit bound go A9 VHH antibody
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's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III. The X-ray crystal structure of A9 bound to RTA (2.6 A resolution) revealed extensive antibody contact with RTA's beta-strand h (732 A2 buried surface area; BSA), along with limited engagement with alpha-helix D (90 A2) and alpha-helix C (138 A2). Collectively, these contacts explain the overlap between epitope clusters I and III, as identified by competition ELISA. However, considerable binding affinity, and, consequently, toxin-neutralizing activity of A9 is mediated by an unusual CDR2 containing five consecutive Gly residues that interact with alpha-helix B (82 A2), a known neutralizing hotspot on RTA. Removal of a single Gly residue from the penta-glycine stretch in CDR2 reduced A9's binding affinity by 10-fold and eliminated toxin-neutralizing activity. Computational modeling indicates that removal of a Gly from CDR2 does not perturb contact with RTA per se, but results in the loss of an intramolecular hydrogen bond network involved in stabilizing CDR2 in the unbound state. These results reveal a novel configuration of a CDR2 element involved in neutralizing ricin toxin. Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity.,Rudolph MJ, Vance DJ, Kelow S, Angalakurthi SK, Nguyen S, Davis SA, Rong Y, Middaugh CR, Weis DD, Dunbrack R Jr, Karanicolas J, Mantis NJ Protein Eng Des Sel. 2018 Jul 1;31(7-8):277-287. doi: 10.1093/protein/gzy022. PMID:30265352[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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