Abstract
Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents. Despite their small size, the high affinity and specificity displayed by VHHs for antigen molecules rival those of IgGs. How such small antibodies achieve that level of performance? Structural studies have revealed that VHHs tend to recognize concave surfaces of their antigens with high shape-complementarity. However, the energetic contribution of individual residues located at the binding interface has not been addressed in detail, obscuring the actual mechanism by which VHHs target the concave surfaces of proteins. Herein, we show that a VHH specific for hen egg lysozyme, D3-L11, not only displayed the characteristic binding of VHHs to a concave region of the surface of the antigen, but also exhibited a distribution of energetic hot-spots like those of IgGs and conventional protein-protein complexes. The highly preorganized and energetically compact interface of D3-L11 recognizes the concave epitope with high shape complementarity by the classical lock-and-key mechanism. Our results shed light on the fundamental basis by which a particular VHH accommodate to the concave surface of an antigens with high affinity in a specific manner, enriching the mechanistic landscape of VHHs.
Highlights
Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents
The kinetic parameters of the interaction between the VHH antibody termed D3-L11 and its antigen hen egg lysozyme (HEL) were determined by surface plasmon resonance (SPR) analysis at 25 °C (Fig. 1a)
We report that the recognition mode of D3-L11 for its antigen HEL shared some features in common with Fv from conventional antibodies[30] and some features in common with other VHHs31,32
Summary
Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents. VHHs comprise only one domain, and for that reason it is less likely that they will adopt the type of large and flat surface displayed by the variable fragments (Fvs) of conventional antibodies Instead, because of their small size, VHHs tend to interact with the concave surface of antigen molecules[17]. It is believed that in this way, VHHs compensate for the limitations of their small size, while maintaining the high affinity and specificity that constitute the hallmarks of antibodies[17] This mechanism has been observed in a structural study that compared the binding mode of VHHs with that of Fvs using hen egg lysozyme (HEL) as a model antigen[18]. We explain the fundamental basis by which D3-L11 accommodate to an antigen cleft with high affinity in a specific manner
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