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 a 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 : l . The association and dissociation at pH 7.4 and 150 mM NaCl had rate constants of 1.9 2 0.3 X 10' M-' s-I and 2 ? 1 X lo-' s-', 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 Eschevichia coli or Chinese hamster ovary cells. The rate of association was only slighly 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 1L-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 (Kd=lOO 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-4IIU-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.

Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

“…Fc-CD95 was biotinylated and coupled with 1 mg/ml to the surface of a streptavidin-coated sensor chip (Pharmacia) as described elsewhere. 24 Sensorgrams were recorded for binding of the various ligands at concentrations ranging from 2 (hCD95L) or 5 nM (mCD95L variants) to 1000 and 5000 nM, respectively. The kinetic constants (k d and k a ) were determined using a 1 : 1 binding model and the software supplied by the manufacturer and were used to calculate the dissociation constant.…”

Prediction of Salt and Mutational Effects on the Association Rate of U1A Protein and U1 Small Nuclear RNA Stem/Loop II

“…The disparate salt effects on k a and k d are observed on many protein-protein pairs. [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48] The binding of U1A with U1SLII shows exactly this salt behavior 7,14 and is thus expected to be modeled by a transient complex close to the native complex.…”

Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry

“…Consequently, there has been great interest in elucidating the mechanisms through which binding partners are able to efficiently recognize each other in solution and adopt the correct conformations, relative positions, and orientations to form native stereo-specific complexes. This has been addressed theoretically (6,7), by computer simulation (8,9), and experimentally (1,5,10,11). The association process is typically broken down into at least two steps.…”

“…The role of electrostatics in binding pathways has been particularly well studied. For a number of systems, including barnase-barstar (5), E9-Im9 (10), IL4-IL4BP (11), and Fas-AchE (1), it has been found that electrostatic stabilization of weakly specific transition states can accelerate association by up to 4 or more orders of magnitude, compared with the basal rate (15). A combination of experimental and computational studies has led to a picture of the transition state in which the binding partners remain solventseparated and interact almost exclusively by long range electrostatics (15,22).…”