Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa 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 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 K(d) for binding at site 2 to be determined. Stopped-flow fluorimetry suggests this to be 3.4 +/- 0.8 microM in good agreement with the value of 4.6 +/- 0.8 microM determined by isothermal titration calorimetry. The mutation Y53F reduces the affinity of site 1 to approximately that of site 2.
Housden
Nicholas G
NG
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, Hants SO16 7PX, United Kingdom.
Harrison
Steven
S
Housden
Hazel R
HR
Thomas
Karen-Anne
KA
Beckingham
Jennifer A
JA
Roberts
Siân E
SE
Bottomley
Stephen P
SP
Graille
Marc
M
Stura
Enrico
E
Gore
Michael G
MG
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010462:Peptostreptococcus; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; C055047:Ig L-binding protein, Peptostreptococcus; D007145:Immunoglobulin kappa-Chains