Global and local determinants for the kinetics of interleukin-4/interleukin-4 receptor alpha chain interaction. A biosensor study employing recombinant interleukin-4-binding protein.
An engineered interleukin-4-binding protein (IL4-BP) representing the extracellular domain of the human interleukin-4 (IL-4) receptor alpha 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 x 10(7) M-1 s-1 and 2 +/- 1 x 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 slightly affected in binding-deficient variants [E9Q]IL-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 degrees C and 37 degrees C yielded similar rates of IL-4 dissociation and only a marginally reduced rate of IL-4 association at 6 degrees C. These results indicate that the high-affinity binding of IL-4 to its receptor (Kd approximately 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.
Shen
B J
BJ
Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) Universität Physiologische Chemie II, Würzburg, Germany.
Hage
T
T
Sebald
W
W
D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015374:Biosensing Techniques; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli; D014644:Genetic Variation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015847:Interleukin-4 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D017354:Point Mutation; D018123:Receptors, Interleukin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D019948:Receptors, Interleukin-4; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D013816:Thermodynamics; D014162:Transfection
D015703:Antigens, CD; D046911:Macromolecular Substances; D018123:Receptors, Interleukin; D019948:Receptors, Interleukin-4; D011994:Recombinant Proteins; D015847:Interleukin-4