Structural evidence for evolution of shark Ig new antigen receptor variable domain antibodies from a cell-surface receptor

Strelsov et al. 10.1073/pnas.0403509101.

Supporting Information

Files in this Data Supplement:

Supporting Table 1
Supporting Figure 6
Supporting Figure 7
Supporting Table 2
Supporting Figure 8

Supporting Figure 6

Fig. 6. Images of σ_A-weighted (2m|F_o |– D|F_c|) difference electron density of the CDR3 loop of the 12Y-2 A chain (a) and B chain (b). The contour level is 1s . The figure was produced by using XTALVIEW and RASTER3D.

Supporting Figure 7

Fig. 7. (a) Alignment of V_NARs 12Y-1 and 12Y-2 with 12 naturally occurring type 2 IgNAR variable domain sequences. Half-cystine residues occur in approximately two-thirds of cases in the CDR1 and CDR3 regions, but are not present in either 12Y-1 or 12Y-2. (b) The 2D topology diagram of 12Y-2 and 12Y-1 folds. The labeled 10 strands form a sandwich of two sheets: front and back sheets are shown with thick (red) and thin (blue) arrows, respectively. The disulfide bond is indicated as an orange rod, the switch of N-terminal strand A to A', adjacent to the bulge in the C-terminal strand G, is shown as a green kink between the A and A' strands.

Supporting Figure 8

Fig. 8. Size-exclusion chromatography of V_NARs 12Y-1 and 12Y-2. (a) Elution profile of affinity-purified 12Y-2 protein on a calibrated Superdex 75 HR10/30 column equilibrated in PBS, pH 7.4, and run at a flow rate of 0.5 ml/min. The peak eluting at 26 min is consistent with a monomeric domain (calculated 12Y-2 M_r of 14,861 Da). The peak eluting at 22 min is consistent with a dimeric protein. The absorbances at A214 nm (unbroken line) and A280 nm (dashed line) are given in arbitrary units. (Inset) The same sample analyzed by SDS/PAGE through a 15% (wt/vol) polyacrylamide Tris/glycine gel and stained with Coomassie brilliant blue. (b) As for a, except the sample analyzed was an isolated 12Y-2 crystal, dissolved in PBS, pH 7.4. A greater proportion of dimeric protein is apparent. (c) As for a, except the 12Y-1 protein was analyzed. Here, the monomer elutes at ≈32 min, and the dimer at ≈28.5 min.