Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype

Abstract

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen-driven affinity-matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an "upright" conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3-specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low-sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen-binding surface.

Figure 1.

Structure of the 12A-9 Type 2 IgNAR antibody variable domain. (A) PyMOL image of the 12A-9 molecule in cartoon representation and colored in rainbow fashion (blue N terminus through to red C terminus). Cysteine residues and disulfide bonds are shown as sticks. (B) XtalView/Raster3D image of σ_A-weighted (2m|F_o|-D|F_c|) electron density of the CDR3 loop of the 12A-9 molecule. The contour level is 1σ. (C) Stereo pair for superimposed structures of IgNAR Type 1, (1SQ2) (cyan); Type 2, 12A-9 (red) and 12Y-2 (1VES) (yellow); and Type 3, modeled (blue) in ribbon representation. Disulphide bonds are shown. (D) PyMol image of superimposed CDR1 loops (22–36) for Type 2 IgNAR’s 12A-9 (red) and 12Y-2 (1VES) (cyan), Type 1 IgNAR (1SQ2) (yellow), and camel cAb-Lys3 (1MEL) (green).

Figure 2.

Modeling of the Type 3 IgNAR antibody isotype. (A) Sequence alignment of Type 3 V_NAR AAM77191 with structural templates used for modeling. The conserved Ig superfamily framework cysteine residues and those found in the Type 2 and Type 3 CDR3 loop regions are highlighted in yellow. Regions of sequence homology are highlighted in gray (green, residues identical in all known V_NAR structures and AAM77191; red, residues conserved in all known V_NAR structures and AAM77191). (B) The 12A-9 CDR loops shown in Cα tube representation. Side-chain atoms of residues involved in packing of the hydrophobic core supporting these loops are shown as cpk balls. Modeled residue side chains for Leu31/Trp31 and Gly96/Phe96 (as well as Ala66/Phe66) are shown in yellow. (C) Predicted model of the Type 3 loop in the same representation as for B. (D) Predicted model of the Type 3 CDR3 loop showing the conserved central hydrophobic Phe96 residue in yellow, surrounded by variable residues putatively involved in antigen binding shown as side-chain atoms rendered as cpk balls. A solvent accessible surface is also shown in gray, orientation ~170° rotation in the vertical axis. (E) Same as for D, but in the same orientation as C, and illustrating the relatively small surface area occupied by the hypervariable residues in the context of the complete single domain antibody. Diagrams were constructed in VMD (Humphrey et al. 1996).