A Collection of Single-Domain Antibodies that Crowd Ricin Toxin's Active Site

In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (V_HHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The V_HHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot ("cluster 3") located in close proximity to RTA's active site. HX-MS analysis revealed that the 21 V_HHs recognized four distinct epitope subclusters (3.1-3.4). Sixteen of the 21 V_HHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three V_HHs grouped within subcluster 3.2, encompassing a-helices C and G, plus α-helix B. The single V_HH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole V_HH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 V_HHs within subclusters 3.1-3.3 physically occlude RTA's active site cleft, while the single antibody in subcluster 3.4 associates on the active site's upper rim.