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-17alpha-hydroxy-progesterone Fab fragment (IgG2b, kappa) was selectively labeled with 13C at carbonyl groups of Ala, Arg, Cys, Ile, Lys, Met, Phe, Trp, or Tyr. The residues in which the carbonyl 13C 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 kappa light chains could crucially affect the interaction between protein L and the V(L) domain.