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.
Wang
Y
Y
Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität, Physiologische Chemie II, Würzburg, Germany.
Shen
B J
BJ
Sebald
W
W
D000409:Alanine / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism*; D001665:Binding Sites; D000939:Epitopes / Q000737:chemistry; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D015847:Interleukin-4 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D018123:Receptors, Interleukin / Q000378:metabolism*; D019948:Receptors, Interleukin-4; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013601:T-Lymphocytes / Q000276:immunology
D015703:Antigens, CD; D000939:Epitopes; D018123:Receptors, Interleukin; D019948:Receptors, Interleukin-4; D011994:Recombinant Proteins; D005973:Glutamine; D015847:Interleukin-4; D000409:Alanine