Structural Characterization of Nanobodies during Germline Maturation

Camelid heavy-chain antibody variable domains (VHH), nanobodies, are the smallest-known functional antibody fragments with high therapeutic potential. In this study, we investigate a VHH binding to hen egg-white lysozyme (HEL). We structurally and dynamically characterized the conformational diversity of four VHH variants to elucidate the antigen-binding process. For two of these antibodies, not only are the dissociation constants known, but also the experimentally determined crystal structures of the VHH in complex with HEL are available. We performed well-tempered metadynamics simulations in combination with molecular dynamics simulations to capture a broad conformational space and to reconstruct the thermodynamics and kinetics of conformational transitions in the antigen-binding site, the paratope. By kinetically characterizing the loop movements of the paratope, we found that, with an increase in affinity, the state populations shift towards the binding competent conformation. The contacts contributing to antigen binding, and those who contribute to the overall stability, show a clear trend towards less variable but more intense contacts. Additionally, these investigated nanobodies clearly follow the conformational selection paradigm, as the binding competent conformation pre-exists within the structural ensembles without the presence of the antigen.

Automated summary

In this study, the conformational diversity of four VHH variants and their binding to hen egg-white lysozyme (HEL) were investigated. It was found that with an increase in affinity, the stability of the VHH structure increased and the residues contributing to antigen binding underwent significant changes. In addition, it was observed that the investigated nanobodies followed the conformational selection paradigm, with the binding competent conformation pre-existing within the structural ensembles without the presence of the antigen.