Mapping binding sites on BR3. (A) Shotgun alanine–scanning mutagenesis of miniBR3 for binding to antibodies and BAFF. The normalized functional ratios (F) observed for each of the scanned positions in BR3 obtained from sequences of positive clones after selection for binding to CB2-IgG, BAFF,³¹  or hybridoma-derived antibodies 9.1, 2.1, and 11G9. Bars with an asterisk above them indicate values that represent a lower limit because alanine was not observed at these positions. (B) Functional epitopes mapped on the structure of hBR3 (1P0T³⁵ ). Distinct antibody determinants (F > 10) based on shotgun alanine–scanning are highlighted for each BR3-binder by coloring corresponding side chains as in panel A; key BAFF-binding residues are circled. Residues that affect a majority of binders are not colored as they likely affect binding indirectly. Residues that differ between hBR3 and mBR3 are underlined. (C) Sequence alignment of the BAFF-binding cysteine-rich domains of hBR3, mBR3, h-BCMA, and h-TACI. Secondary structure and disulfide-bonding pattern of BR3 are shown. Residues identical to hBR3 are shaded. The conserved DxL(V/L) motif is boxed.