Comparative analysis of nanobody sequence and structure data

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.

Keywords: HcAb; VH; VHH; antibody; camelid; framework; heavy chain antibody; loop; single domain antibody.

Figure 1

Structural features of conventional and camelid heavy‐chain Abs. A, In an Ab, the antigen binds to the VH‐VL interface, while in the camelid heavy‐chain antibody the VH‐homologous VHH domain binds the antigen. Nbs are produced when the VHH domain is expressed in bacterial systems. In Abs, the VH and VL domains bind to each other and can only be produced in bacterial expression systems when joined by a peptide linker. B, Like the Ab VH domain, the secondary structure of the Nb VHH domain consists of 9 beta sheets separated by loop regions, 3 of which are hypervariable (shown in blue, green and red). Four framework regions (FRs) separate the variable loops; these are less sequence‐variable. Four positions known as the VHH‐tetrad are numbered and highlighted in yellow. Right: VHH domain with VHH‐tetrad positions in yellow. C, The antigen‐binding surface in Nb VHH domains and Ab VH‐VL domains (aligned orientations) [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

A, Summary of sequence variability in sets of Nb VHH domain and B, Ab VH domain sequences. The number of sequences with a residue (ie, not a gap character) at each alignment position is plotted in dark blue. The number of sequences with the most frequent amino acid at that position (nWT) is overlaid in magenta or cyan. The central horizontal bar indicates the framework (gray) and loop regions (colored). Despite comparable diversity of epitope specificities among the two datasets we note that the framework regions of the Nb VHH domains are significantly more conserved than those of the Ab VH domains. Those alignment positions that are involved in the VH‐VL interface, and the equivalent positions in VHH domains, are marked in yellow [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

A, Sequence logo plots that show the amino acid variation present in the Nb VHH domains and B, Ab VH domains from the datasets described. Aligned sequence positions with high propensity (> 10% of structures) to contact the antigen are marked in orange. Note the difference in residue composition between VH and VHH at the four VHH‐tetrad positions 37, 44, 45, and 47. Our alignments show VHH position 47 can frequently be Phe, Leu or Trp in addition to the often cited Gly. It is also interesting that the two VHH charged residues at positions 44 and 45 are frequently in contact with the antigen, rather than with the VL, as they are in VH‐VL complexes [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 4

Comparison of Nb structural diversity to Ab structural diversity. Superposition of A, 90 Nb VHH domains and B, 90 Ab VH‐VL domains from co‐crystal structures. Front, back and plan views, with H1 in blue; H2 in green and H3 in red. The VHH and VH domains are aligned in each of the three views shown

Figure 5

Sequence and structural analysis of Nb hypervariable loops H1–3. We characterize amino acid usage, loop length distribution, sequence diversity and structural diversity of Nb A, H1 loops, B, H2 loops, and C, H3 loops. Central loop positions with more than 85% aligned gap characters, which were excluded from the 126‐position alignment, are highlighted in yellow. Framework “anchors,” highlighted in gray, can be used to detect the loop locations in individual sequences19 [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Sequence and structural analysis of Ab VH domain hypervariable loops H1–3. We characterize amino acid usage, loop length distribution, sequence diversity and structural diversity of Ab A, H1 loops, B, H2 loops, and C, H3 loops. Central loop positions with more than 85% aligned gap characters, which were excluded from the 126‐position alignment, are highlighted in yellow. Framework “anchors,” shown in gray, are more variable than in the VHH alignment, but can still be used to detect the loop locations in individual sequences [Color figure can be viewed at http://wileyonlinelibrary.com]