The high-affinity interaction of human IL-4 and the receptor alpha chain is constituted by two independent binding clusters.
IL4-BP, the extracellular binding domain of the IL-4 receptor alpha chain, contains a high-affinity binding epitope for IL-4 (K(D) 150 pM). Previous results on the crystal structure of the IL-4/IL4-BP complex and on the functional epitope of IL-4 suggested that this contact comprises a mosaic of two binding clusters. The present mutational analysis of IL4-BP supports this view and demonstrates that the energetically most important group is the receptor carboxylate group of D72 forming an ion pair with IL-4 R88 in cluster II. The second main receptor determinant is the hydroxyl group of Y183 forming a hydrogen bond with IL-4 E9 in cluster I. The latter is engaged in additional hydrogen bonds with Y13 and also in van der Waals contacts with Y127. Receptor residue D72 as well as Y183 are each surrounded by a shell of hydrophobic groups from residues that upon mutation lead to smaller decreases in binding affinity. Analysis of IL4-BP double mutants showed that receptor side-chains within one cluster but not those of different clusters cooperate. Interaction analysis of IL-4 and IL4-BP single mutants also revealed additivity in binding of side-chains between clusters and cooperativity within each cluster I or II.These results show that the high-affinity IL-4/IL4-BP contact is constituted by two independent binding units, each containing a central polar or charged side-chain surrounded by hydrophobic groups (avocado cluster).
Zhang
Jin-Li
JL
Department of Physiological Chemistry II, Biocenter, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany.
Simeonowa
Irina
I
Wang
Yonghong
Y
Sebald
Walter
W
D001665:Binding Sites; D018360:Crystallography, X-Ray; D000939:Epitopes; D006801:Humans; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D015847:Interleukin-4 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D019948:Receptors, Interleukin-4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D000939:Epitopes; D008024:Ligands; D019948:Receptors, Interleukin-4; D015847:Interleukin-4