Direct antibody-inhibition of GpA binding by RII.

<p>(A) RII binds to GpA (lane 1), but not to neuraminidase treated GpA (GpA-NA – lane 2). Addition of R217 IgG or Fab prevents RII from binding GpA (lane 3 and 4). Neither R218 IgG (lane 5), R218 Fab (lane 6), control IgG (lane 7) nor control Fab (lane 8), block RII/GpA receptor binding. Binding is specific as neuraminidase treatment prevents binding in all cases (lanes 2 and 9–14). Note that an increased signal is observed with R218 IgG over R218 Fab (compare lanes 5 and 6) suggesting bivalent binding. This assay was performed with RII at 3 µM and antibody or Fab fragments at 6 µM. (B) Titration of R217 demonstrates the specificity of interaction as all concentrations around and above the available RII concentration (3 µM) prevents binding (lanes 6–10). At concentrations below the available RII binding occurs as not all the RII is bound by R217 (lanes 3–5). (C) Titration of R218 demonstrates that R218 is unable to directly prevent GpA binding at any concentration.</p

Antibody epitope identification by immunofluorescence assay.

<p>DBL domain proteins were surface expressed on HEK293 cells and probed with R217 or R218 as primary and Alexafluor-546 labeled anti-IgG₁ as secondary. Green channel shows GFP tagged expressed protein. Red channel shows Alexafluor-546 labeled proteins on HEK293 cell surface. Merged channel shows overlap between green and red channels.</p

Crystal structure of RII/R217 Fab complex.

<p>(A) Overall structure of the RII/R217 Fab complex shown in ribbon representation. The F1 domain of RII is colored in green, the F2 domain of RII is colored purple. The Fab heavy chain (VH) is in blue and the light chain (VL) in pink. The location of F2 β-finger is circled in black. (B) Ribbon representation of F2 mapping the R217 epitope. Residues contacted by the Fab are show in stick. Residues contacted by the heavy chain are colored blue, residues contacted by the Fab light chain are colored pink, and residues contacted by both chains are in beige. Residues not contacted by the antibody are in purple. (C) Surface representation of F2 mapping the R217 epitope. Color scheme as in B. (D) Surface representation of the R217 Fab, mapping heavy chain residues (blue) that contact F2 (purple). The light chain is shown in white. (E) Surface representation of the R217 Fab, mapping light chain residues (pink) that contact F2 (purple). The heavy chain is shown in white.</p

Crystal structure of F1/R218 Fab complex.

<p>(A) Overall structure of the F1/R218 Fab complex shown in ribbon representation. The F2 domain of RII is colored in green. The Fab heavy chain (VH) is in blue and the light chain (VL) in pink. (B) Ribbon representation of F1 mapping the R218 epitope. Residues contacted by the Fab are show in stick. Residues contacted by the heavy chain are colored blue, residues contacted by the Fab light chain are colored orange, and residues contacted by both chains are in beige. Residues not contacted by the antibody are in green. (C) Surface representation of F2 mapping the R217 epitope. Color scheme as in B. (D) Surface representation of the R218 Fab, mapping heavy chain residues (blue) that contact F2 (green). The light chain is shown in white. (E) Surface representation of the R218 Fab, mapping light chain residues (orange) that contact F2 (green). The heavy chain is shown in white.</p