A mixed-charge pair in human interleukin 4 dominates high-affinity interaction with the receptor alpha chain.

Human interleukin 4 (IL-4) binds to its cellular receptor with a Kd in the subnanomolar range, similar to many other 4-helix-bundle proteins interacting with members of the hematopoietin (cytokine) receptor superfamily. In the IL-4 system this interaction is predominantly determined by the extracellular domain (IL4-BP) of the receptor alpha chain (Kd approximately 150 pM). Now a high-resolution mutational and kinetic analysis has revealed that the high-affinity binding of IL-4 originates from a continuous patch of a few mostly polar or charged amino acid side chains located on helices A and C. The binding epitope comprises (i) a set of side chains determining the dissociation rate (k(off)) and (ii) a partially overlapping set determining the association rate constant (k(on)) of the IL-4/IL4-BP complex. The k(off) epitope is assembled from two juxtaposed main determinants (Glu-9 and Arg-88) surrounded by five side chains (Ile-5, Thr-13, Arg-53, Asn-89, and Trp-91) of lower importance. The cumulative increase in k(off) after alanine substitution is 10(5)-fold for the central mixed-charge pair and 3 x 10(3)-fold for the satellites. The k(on) epitope is formed by five positively charged residues on helix C (Lys-77, Arg-81, Lys-84, Arg-85, and Arg-88) and two neighboring residues on helix A (Glu-9 and Thr-13). The cumulative loss in k(on) of the alanine variants is only about 10-fold. These results provide the basis for an understanding of molecular recognition in cytokine receptor complexes and for an IL-4 antagonist design.