Abstract
Nanobodies (Nbs) are a class of antigen-binding protein derived from camelid immune systems, which achieve equivalent binding affinities and specificities to classical antibodies (Abs) despite being comprised of only a single variable domain. Here, we use a data set of 156 unique Nb:antigen complex structures to characterize Nb–antigen binding and draw comparison to a set of 156 unique Ab:antigen structures. We analyse residue composition and interactions at the antigen interface, together with structural features of the paratopes of both data sets. Our analysis finds that the set of Nb structures displays much greater paratope diversity, in terms of the structural segments involved in the paratope, the residues used at these positions to contact the antigen and furthermore the type of contacts made with the antigen. Our findings suggest a different relationship between contact propensity and sequence variability from that observed for Ab VH domains. The distinction between sequence positions that control interaction specificity and those that form the domain scaffold is much less clear-cut for Nbs, and furthermore H3 loop positions play a much more dominant role in determining interaction specificity.
Highlights
Nanobodies (Nbs) are small single-domain proteins found in the immune systems of camelid species (Hamers-Casterman et al, 1993)
The Nb consists of the VHH domain from the homodimeric camelid heavy chain antibody (HcAb), which is homologous to the Ab heavy chain variable domain (VH domain)
We analyse the antigen-contacting residues from two sets of Nb:antigen and Ab: antigen co-crystal structures, to probe how paratopes are constructed and how they differ between the two classes of Ab
Summary
Nanobodies (Nbs) are small single-domain proteins found in the immune systems of camelid species (Hamers-Casterman et al, 1993). Isolated VHH domains are highly soluble and are fast emerging as an alternative Ab fragment for therapeutic, diagnostic and molecular research applications (De Meyer et al, 2014). Nbs are thought to be functionally equivalent to full-length classical Abs; achieving nanomolar binding affinities and high specificities to seemingly any antigen the camelid immune system is challenged with (Muyldermans, 2013). These binding capabilities are achieved using a single variable domain with just three sequence-variable loops, compared to two variable domains of the VHVL binding subunit from monoclonal Abs (see Fig. 1). Nbs are distinguished by four critical framework mutations that enhance solubility, and H3 loops that are around 15% longer than Ab H3 loops (Sircar et al, 2011; Muyldermans, 2013; Mitchell and Colwell, 2018)
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