Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies

Abstract

In this report we investigated, within a group of closely related single domain camelid antibodies (VHHs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast-acting toxin and biothreat agent. The V1C7-like VHHs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin-neutralizing activities. Using the X-ray crystal structure of V1C7 in complex with ricin’s enzymatic subunit (RTA) as a template, Rosetta-based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7’s binding affinity for ricin, whereas the reverse (i.e., Gly for Arg at position 29) diminished V5C1’s binding affinity by >10 fold. As expected, the V5C1R29G mutant was largely devoid of toxin-neutralizing activity. However, the toxin-neutralizing activity of the V1C7G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen-deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

“…The relationship between binding affinity and TNA was most apparent within V H H clonal families (Fig. 2C), the members of which would be expected to have epitopes similar to each other (31). The failure of antibodies such as JNM-B5 to neutralize ricin despite its relatively high binding affinity (e.g., 60 pM) demonstrates once again that other factors such as epitope specificity contribute to antibody functionality.…”

“…PA1 and SyH7 contact the loop before ␣-helix A and ␣-helices F and G, while PH12, TB12, and SWB1 contact ␤-strand d and ␣-helices D and E. Moreover, as noted above, the structural epitopes of two V H Hs in bin 7, JIY-E1 and V1C7, (Table 3). Rosetta-based homology modeling suggests that the V1C7 family members, despite their minor differences in CDR sequences, interact with RTA in similar fashions (31). Thus, the epitopes of all five V H Hs in bin 7 can be positioned on RTA with relative accuracy.…”

“…6). To refine these ambiguous antibody binding sites, we subjected V1B11 and JNM-D1 to epitope mapping by hydrogendeuterium exchange mass spectrometry (HX-MS), as described recently (31). HX-MS affords peptide-level resolution of antibody binding sites and is therefore an extremely useful methodology for epitope localization (33).…”

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

“…A reduction in HX across a peptide is interpreted as protection due to protein-protein contacts and, therefore, diagnostic of epitopic regions (45). The magnitude of this protection was quantified using the deuteration difference normalized by maximal deuteration as follows: , and were applied successfully to V H H-RTA interactions, where we compared the HX-MS output to a known X-ray crystal structure (51). The magnitudes of altered HX were classified by k-means clustering into four categories and were color coded accordingly as follows: strong protection, deep blue; intermediate protection, light blue; no protection, gray; deprotection, yellow.…”

“…Hydrogen exchange-mass spectrometry (HX-MS) was the method of choice based on recent success in a wide variety of pathogens, including HIV, the malaria parasite, Neisseria meningitidis, and Staphylococcus aureus (45)(46)(47)(48)(49)(50). In a recent study, we successfully adapted HX-MS for the purpose of identifying epitopes recognized by a family of RTA-specific single-domain alpaca-derived antibodies (V H Hs) (51). We also used HX-MS to assist in epitope refinement of a subset of the 68 V H Hs described in a companion paper (71).…”