Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains.

Nicholas G Housden,Enrico Stura,Stephen P Bottomley,Jennifer A Beckingham,Siân E Roberts,Marc Graille,Hazel R Housden,Karen-Anne Thomas,Michael G Gore,Steven Harrison

doi:10.1074/jbc.m312938200

Abstract

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.

Highlights

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 ␬ light chain are due to the binding of the ␬ light chain at two separate sites on the protein L molecule
The residual fluorescence intensity at equilibrium given by Y64W/ Y53F is more than half of the fluorescence intensity difference given by Y64W and Y64W/Y53F/L57H
This suggests that the Y64W/Y53F has an affinity for ␬-chain at site 1 that is lower than its affinity at site 2

Summary

The abbreviations used are

PpL312, single Ig-binding domain of protein L from strain 312 of P. magnus; PpL3316, single Ig-binding domain of protein L from strain 3316 of P. magnus; ␬-chain, ␬ light chain; wt, wild-type; FRET, fluorescence resonance energy transfer. Previous enzyme-linked immunosorbent assay experiments (11) have shown that nitration of Tyr[53] or its mutation to Phe dramatically increases the Kd of the ␬-chainPpL3316 interaction Another important residue at site 1 is Leu[57]. The mutation D55A eliminates a salt bridge and dramatically weakens or eliminates binding (12), and the mutation A66W is predicted to cause a steric clash removing binding at site 2 This is born out by fluorimetry experiments reported here and the A66W PpL3316 is suitable for determining the affinity of site 1. The I34W PpL3316 mutant, which has a Trp residue located toward the N terminus of ␤-strand 2, has been selected to study binding at the site 1 interface (see Fig. 1a); this construct is tolerant to the introduction of further mutations.

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

15 Ϯ 2 15b

CONCLUSIONS