Global and Local Determinants for the Kinetics of Interleukin‐4/Interleukin‐4 Receptor α Chain Interaction

Abstract

An engineered interleukin‐4‐binding protein (IL4‐BP) representing the extracellular domain of the human interleukin‐4 (IL‐4) receptor α chain was expressed in Sf9 cells. The purified IL4‐BP was immobilized via a single biotinylated SH group near the carboxyl end to a biosensor matrix and analysed in real time for interaction with IL‐4 and IL‐4 variants. IL‐4 was bound to IL4‐BP at a molar ratio of approximately 1:1. The association and dissociation at pH 7.4 and 150 mM NaCl had rate constants of 1.9 ± 0.3 × 10 7 M −1 s −1 and 2 ± 1 × 10 −3 s −1 , respectively. Glycosylation and engineered amino acid substitutions of IL4‐BP did not alter the kinetic constants as shown by a parallel analysis of IL4‐BP variants produced in Escherichia coli or Chinese hamster ovary cells. The rate of association was only slighly affected in binding‐deficient variants [E9QJIL‐4 and [R88Q]IL‐4 and by acidic pH down to values of 4.5, but it was reduced up to fivefold at higher ionic strength. The rate of dissociation was increased 70‐fold and 150‐fold with the IL‐4 variants and fivefold at an acidic pH of 4.5, but it was not affected by high ionic strength. Temperatures between 6°C and 37°C yielded similar rates of IL‐4 dissociation and only a marginally reduced rate of IL‐4 association at 6°C. These results indicate that the high‐affinity binding of IL‐4 to its receptor ( K d ≈100 pM) is mainly the result of an unusually high association rate. The IL‐4/IL4‐BP interaction appears to be dominated by charge effects. The exceedingly high rate of IL–4/IL4‐BP association is augmented by the overall electrostatic potentials of both proteins (electrostatic steering). Localized charges and the formation of ion pairs may control the rate of complex dissociation.