Housden, N.G., Harrison, S., Housden, H.R., Thomas, K.-A., Beckingham, J.A., Roberts, S.A., Bottomley, S.P., Graille, M., Stura, E. and Gore, M.G.
Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains.
Journal of Biological Chemistry, 279, (10), . (doi:10.1074/jbc.M312938200).
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 kappa light chain are due to the binding of the kappa 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 kappa 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 Kd for binding at site 2 to be determined. Stopped-flow fluorimetry suggests this to be 3.4 +/- 0.8 muM in good agreement with the value of 4.6 +/- 0.8 muM determined by isothermal titration calorimetry. The mutation Y53F reduces the affinity of site 1 to approximately that of site 2.
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