Antibodies that recognize small molecule ligands (haptens) provide unique insight into the immune response and frequently serve as biological reagents for the detection of small molecules. While conventional antibodies typically recognize haptens using two variable domains (VL and VH), much less is known regarding how antibodies with a single variable domain recognize small ligands. Here we investigate the binding thermodynamics for an anti-caffeine camelid (VHH) antibody. Surprisingly, a nonconventional binding stoichiometry was observed in which the final complex includes two VHH domains for every caffeine molecule. DeltaC(p) analysis and size exclusion chromatography support this unusual stoichiometry. An apparent consequence of ligand-induced dimerization is that a relatively high affinity (K(b,obs) = 7.1 x 10(7)) is obtained. The binding profiles of three caffeine metabolites, theophylline, theobromine, and paraxanthine, were also investigated. Each ligand maintains a 2:1 stoichiometry while displaying an approximately 50-fold range of observed binding affinities. These results suggest nonconventional mechanisms of hapten recognition are possible with single-domain antibodies.
Camelid heavy-chain-only antibodies are a unique class of antibody that possesses only a single variable domain (termed VHH) for antigen recognition. Despite their apparent canonical mechanism of target recognition, where a single VHH domain binds a single target, an anti-caffeine VHH has been observed to possess 2:1 stoichiometry. Here, the structure of the anti-caffeine VHH/caffeine complex enabled the generation and biophysical analysis of variants that were used to better understand the role of VHH homodimerization in caffeine recognition. VHH interface mutants and caffeine analogs, which were examined to probe the mechanism of caffeine binding, suggested caffeine recognition is only possible with the VHH dimer species. Correspondingly, in the absence of caffeine, the anti-caffeine VHH was found to form a dimer with a dimerization constant comparable to that observed with VH:VL domains in conventional antibody systems, which was most stable near physiological temperature. While the VHH:VHH dimer structure (at 1.13 Å resolution) is reminiscent of conventional VH:VL heterodimers, the homodimeric VHH possesses a smaller angle of domain interaction, as well as a larger amount of apolar surface area burial. To test the general hypothesis that the short complementarity-determining region-3 (CDR3) may help drive VHH:VHH homodimerization, an anti-picloram VHH domain containing a short CDR3 was generated and characterized, which revealed it also existed as dimer species in solution. These results suggest homodimer-driven recognition may represent a more common method of VHH ligand recognition, opening opportunities for novel VHH homodimer affinity reagents and helping to guide their use in chemically induced dimerization applications.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.