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(H)Hs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The V(H)Hs, 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(H)Hs recognized four distinct epitope subclusters (3.1–3.4). Sixteen of the 21 V(H)Hs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three V(H)Hs grouped within subcluster 3.2, encompassing α-helices C and G, plus α-helix B. The single V(H)H in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole V(H)H 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(H)Hs 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.