NMR analysis of the interaction between protein L and Ig light chains

Protein L is a cell wall protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. It binds to immunoglobulin (Ig) Light chains predominantly of the kappa subtype from a wide range of animal species. This binding is mediated by five highly homologous repeats designated as B1-B5, each of which comprises 72 to 76 amino acid residues. The fold of the Ig light chain-binding B1 domain of protein L has previously been shown to comprise an alpha-helix packed against a four-stranded beta-sheet. The Ig-binding region of the protein L domain involves most of the residues in the second beta-strand, the C-terminal residues of the alpha-helix, and residues in the loop connecting the alpha-helix with the third beta-strand. in the present study, we have identified the protein L-binding site of an Ig light chain by use of stable isotope-assisted NMR spectroscopy. The light chain of a murine monoclonal anti-17 alpha-hydroxyprogesterone Fab fragment (IgG2b, kappa) was selectively labeled with C-13 at carbonyl groups of Ala, Arg, Cys, Ile, Lys, Met, Phe, Trp, or Tyr. The residues in which the carbonyl C-13 chemical shift was significantly perturbed upon binding of the protein L B1 domain were preferentially found in the second beta-strand of the variable kappa domain and parts of its flanking beta-strands. None of these residues were affected by the addition of the antigen against which the monoclonal Fab fragment is directed. Therefore, we conclude that protein L binds to the outer surface of the framework region of the V-L domain, primarily involving the V-L second strand, and that this binding is independent of antigen-binding. The present NMR data, in combination with sequence comparisons between kappa light chains with and without protein L affinity, suggest that the amino acid substitutions at positions 9, 20, and/or 74 of the Ic light chains could crucially affect the interaction between protein L and the V-L domain. (C) 1997 Academic Press Limited.