Comparative analysis of nanobody sequence and structure data

TLDR

Nanobodies are camelid-derived, single-domain antibodies that bind antigens with high affinity and specificity, yet their small size raises questions about how they achieve such precise binding. The study aims to determine how nanobodies achieve high specificity by comparing 90 nanobody–antigen crystal structures to a matched set of classical antibodies. We compiled 90 nonredundant nanobody–antigen crystal structures and an analogous set of classical antibody structures to analyze sequence and structural determinants of specificity. Nanobodies achieve specificity not by diversifying their framework but by expanding paratope sequence positions and increasing structural variability in H1, H2, and H3 loops.

Abstract

Nanobodies are a class of antigen-binding protein derived from camelids that achieve comparable binding affinities and specificities to classical antibodies, despite comprising only a single 15 kDa variable domain. Their reduced size makes them an exciting target molecule with which we can explore the molecular code that underpins binding specificity-how is such high specificity achieved? Here, we use a novel dataset of 90 nonredundant, protein-binding nanobodies with antigen-bound crystal structures to address this question. To provide a baseline for comparison we construct an analogous set of classical antibodies, allowing us to probe how nanobodies achieve high specificity binding with a dramatically reduced sequence space. Our analysis reveals that nanobodies do not diversify their framework region to compensate for the loss of the VL domain. In addition to the previously reported increase in H3 loop length, we find that nanobodies create diversity by drawing their paratope regions from a significantly larger set of aligned sequence positions, and by exhibiting greater structural variation in their H1 and H2 loops.