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. 2018 Jul 1;31(7-8):267-275.
doi: 10.1093/protein/gzy017.

Analysis of nanobody paratopes reveals greater diversity than classical antibodies

Laura S Mitchell  1 , Lucy J Colwell  1
Affiliations

Analysis of nanobody paratopes reveals greater diversity than classical antibodies

Laura S Mitchell et al. Protein Eng Des Sel. .

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.

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Figures

Fig. 1
Fig. 1
Schematic overview of VHH and VH domains. VHHs (Nbs) are the variable domain derived from camelid HcAbs (A), whereas VHs are the heavy chain variable domain derived from classical Abs (B). These homologous immunoglobulin domains have four key differences, represented in (C): (i) Nb H1 loops tend not to fit canonical conformations seen in Ab H1 loops, (ii) Nb H3 loops can be longer in length, (iii) Nb framework regions (FRs) are more conserved in both sequence and structure than Ab FRs and (iv) Nb FR2 have four solubility-enhancing mutations that facilitate stability without a partnering VL domain.
Fig. 2
Fig. 2
Variable domains in Abs can be conceptually divided into two regions. Structurally, the domain may be divided into framework and loop regions; or functionally it may be considered in terms of specificity-determining regions and scaffold regions (A). These distinctions are interchangeable in Abs, since the sequence-variable loop regions coincide with antigen contacts. This distinction is useful for (B) predicting paratopes from Ab sequences, (C) modelling Ab:antigen interactions and (D) engineering Ab properties.
Fig. 3
Fig. 3
Contact profiles that describe the combination of interface residues used to contact the antigen by (A) Nbs and (B) Ab VH domains. The central dot matrix represents the range of observed contact profiles. Each profile is denoted by a vertical strip of dots, which indicates the combination of structural segments involved in a paratope. For example, the second profile with a blue, green and red dot indicates the class of Nbs and Abs that bind the antigen using only residues from loops H1–3. The frequency of each contact profile in the data set is indicated by the butterfly format bar charts. Note that while Abs often use just the three hypervariable loops H1–3 to contact the antigen (83/156), Nbs use a much more diverse distribution of possible contact profiles.
Fig. 4
Fig. 4
Antigen contact frequency for each of the 126 aligned positions in our data sets of Nb (A) and Ab (B) co-crystal structures, shown alongside the difference in mean SASA per aligned position (C). In (A) and (B), the number of sequences with a residue at each alignment position is plotted in light purple, and the number of structures that contact the antigen at each alignment position is overlaid in dark purple. Note the increased frequency of framework residues that contact the antigen in Nb VHH domains compared to Ab VH domains, which is linked to increased SASA in Nbs at these positions. The full distribution of SASAs for each aligned position, from which the difference in mean SASA was calculated is provided in Supplementary Fig. S3, data available at PEDS online.
Fig. 5
Fig. 5
Scatter plots showing the relationship between sequence variability and contact propensity for each position of the AHo alignment, for (A) Nbs and (B) Ab VH domains. Points are labelled with alignment position, coloured according to structural segment of the molecule and sized according to the number of sequences with a residue at the alignment position. Inset representative Nb and Ab structures with surface rendering are oriented equivalently, and underlying residues are coloured according to the specificity-determining (purple), intermediate (yellow) or scaffold (grey) clusters they belong to. In the lower panel (C), the difference in conservation and contact propensity between equivalent VHH and VH alignment positions is plotted. A key to alignment positions and clusters is provided in the lower right panel.
Fig. 6
Fig. 6
Heatmaps of the frequency of pairwise residue–residue contacts across Nb:antigen (left) and Ab:antigen (right) interfaces. Larger heatmaps (A) and (B) represent the frequency of each pairwise contact type across the entire full-length alignments, with high-frequency pairings coloured purple, and low-frequency pairings coloured white. Contacts contributed from the Nb or Ab are plotted along x-axis and contacts contributed from antigen are plotted along the y-axis. Histograms show the distribution of contacting residue type contributed by the two halves of the interface, with percentages annotated for the Nb/Ab side of the interface. Smaller heatmaps (C) and (D) represent a breakdown of pairwise residue–residue contact frequencies contributed by structural segments H1, H2, H3 and all four FRs combined. Total number of pairwise contacts represented by each plot is given in the titles. Equivalent plots for VH and VL domains are in Supplementary Fig. S5, data available at PEDS online.
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References

    1. Abhinandan K.R. and Martin A.C.R. (2008) Mol. Immunol., 45, 3832–3839. - PubMed
    1. Adolf-Bryfogle J., Xu Q., North B., Lehmann A. and Dunbrack R.L. (2015) Nucleic Acids Res., 43, D432–D438. - PMC - PubMed
    1. Berman H.M., Westbrook J., Feng Z., Gilliland G., Bhat T.N., Weissig H., Shindyalov I.N. and Bourne P.E. (2000) Nucleic Acids Res., 28, 235–242. - PMC - PubMed
    1. Chakravarty R., Goel S. and Cai W. (2014) Theranostics, 4, 386–398. - PMC - PubMed
    1. Chothia C., Lesk A.M., Tramontano A. et al. . (1989) Nature, 342, 877–883. - PubMed

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