Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity
- 1 July 2018
- journal article
- research article
- Published by Oxford University Press (OUP) in Protein Engineering, Design and Selection
- Vol. 31 (7-8), 277-287
- https://doi.org/10.1093/protein/gzy022
Abstract
Ricin 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 angstrom resolution) revealed extensive antibody contact with RTA's beta-strand h (732 angstrom(2) buried surface area; BSA), along with limited engagement with alpha-helix D (90 angstrom(2)) and alpha-helix C (138 angstrom(2)). 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 angstrom(2)), 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.Keywords
Funding Information
- National Institutes of Allergy and Infectious Diseases
- National Institutes of Health (GM122817)
- National Institute of General Medical Sciences
This publication has 44 references indexed in Scilit:
- Plant-based expression of a partially humanized neutralizing monoclonal IgG directed against an immunodominant epitope on the ricin toxin A subunitVaccine, 2011
- Rosetta3Methods in Enzymology, 2010
- A New Clustering of Antibody CDR Loop ConformationsJournal of Molecular Biology, 2010
- Folding domains within the ricin toxin A subunit as targets of protective antibodiesVaccine, 2010
- PyRosetta: a script-based interface for implementing molecular modeling algorithms using RosettaBioinformatics, 2010
- RiVax, a recombinant ricin subunit vaccine, protects mice against ricin delivered by gavage or aerosolVaccine, 2007
- Phasercrystallographic softwareJournal of Applied Crystallography, 2007
- ARP⧸wARP and Automatic Interpretation of Protein Electron Density MapsMethods in enzymology, 2003
- The Protein Data BankNucleic Acids Research, 2000
- [20] Processing of X-ray diffraction data collected in oscillation modeMethods in Enzymology, 1997