Observation and Characterization of the Interaction between a Single Immunoglobulin Binding Domain of Protein L and Two Equivalents of Human κ Light Chains

Detailed stopped-flow studies in combination with site-directed mutagenesis, isothermal titration calorimetry data and x-ray crystallographic knowledge have revealed that the biphasic pre-equilibrium fluorescence changes reported for a single Ig-binding domain of protein L from Peptostreptococcus magnus binding to κ light chain are due to the binding of the κ light chain at two separate sites on the protein L molecule. Elimination of binding site 2 through the mutation A66W has allowed the K_d for κ light chain binding at site 1 to be measured by stopped-flow fluorescence and isothermal titration calorimetry techniques, giving values of 48.0 ± 8.0 nM and 37.5 ± 7.3 nM respectively. Conversely, a double mutation Y53F/L57H eliminates binding at site 1 and has allowed the K_d for binding at site 2 to be determined. Stopped-flow fluorimetry suggests this to be 3.4 ± 0.8 μM in good agreement with the value of 4.6 ± 0.8 μM determined by isothermal titration calorimetry. The mutation Y53F reduces the affinity of site 1 to approximately that of site 2.