The interleukin-4/receptor α chain complex (PDB ID: 1IAR) is a regulatory cytokine that is produced by the T helper type 2 (Th2) cells of the immune system. More generally, cytokines are proteins that act as intracellular signals; they travel in the bloodstream and bind to a target cell to alter the cell’s behavior. They function by binding with high affinity to a cytokine-binding chain, designated “α,” and then interacting with lower affinity to a receptor chain on a cell surface, such as the common γ chain (γc) or IL-13Rα1 (3). The receptor chains vary in number and proportion on different types of cells (3).
Interleukins are cytokines with various immune system-related roles. They activate T and B lymphocytes, stimulate growth and maturation of these cells, and mediate inflammation and antibody secretion. IL-4 is one of fifteen interleukins, designated numerically IL-1 through IL-15. Since the IL-4/IL4-BP complex can bind to two different types of receptors, it has both regulatory and effector functions. Regulatory functions of IL-4 include Th2 differentiation (and Th2 cells also secrete other interleukins and factors that raise eosinophil production in the bone marrow and B-cell differentiation into plasma cells [1]), immunoglobulin class switching, dendritic cell maturation, and macrophage activation (3). Effector functions of IL-4 include airway hypersensitivity (bronchoconstriction), goblet cell metaplasia, which is the increased differentiation of bronchial epithelial cells into mucus-secreting goblet cells, tissue eosinophilia, and other structural changes in the airway (called airway remodeling) associated with chronic asthma (1). However, IL-4 plays a lesser role in effector functions because IL-13 (which also binds to the α chain complex) is much more numerous and IL-13 is the cytokine primarily responsible for those effects (3). Though IL-4 and IL-13 normally exist in all humans, certain polymorphisms lead to defects in the immune system triggering pathway, leading to immunological or inflammatory diseases (1).
IL-4 binds tightly with the interleukin-4 binding protein (IL4-BP) to form a complex that recruits the common γ chain (γc). The binding of γc allows the complex to activate the transmembrane signaling responsible for the Th2-mediated immune response associated with allergic and inflammatory diseases (1, 2). IL-2, IL-7, IL-9, and IL-15 are also cytokines that recruit γc. This entire family is responsible for the activation of Immunoglobulin E-producing B cells, eosinophils, and mast cells necessary for launching an immune response against parasites in mucosal tissues and the skin (2).
Physical parameters of each protein were found using ExPASy. IL-4 has 129 residues, a molecular weight of 14963.2 Daltons, and a theoretical isoelectric point (pI) of 9.26. IL4-BP has 207 residues, a molecular weight of 23716.6 Daltons, and a theoretical pI of 5.77 (6). The Protein Database reveals that the secondary structure of IL-4 is composed of 62% helices and 4% beta sheets, and IL4-BP is composed of 6% helices and 44% beta sheets (8).
IL4-BP has two domains that are covalently linked to form an L-shape. This “sandwich”-type formation comprises a total of seven antiparallel β sheets, divided into a three-strand and four-strand β-pleated sheet. IL-4 comprises three helices. The binding epitope on IL-4 is composed of three trans-interacting clusters, two of which bind with high affinity for IL4-BP, and one of which has merely a “steering” role by virtue of electrostatic interactions that stabilize the complex (2). The two “avocado clusters” that bind with high affinity to the receptor have an amphipathic structure involving an outer cloak of hydrophobic residues surrounding a core of polar or charged residues. The amphipathic structure seems to be crucial to the high affinity between the two clusters and the receptor because polar interactions are stronger in a hydrophobic environment (2).
Binding between the IL-4 and IL4-BP occurs at cluster I and cluster II. In cluster I of IL-4, E9 forms a net of hydrogen bonds with a set of three tyrosine residues in the receptor. The most important hydrogen bond from E9 is formed with the hydroxyl group of Y183 in the receptor. It forms additional hydrogen bonds with Y13 and has van der Waals interactions with Y127 in the receptor, though these interactions play a lesser role in contributing to the stability of the complex. In cluster II of IL-4, R88 forms an ionic bond with the carboxylate group of receptor D72 (4). IL-4’s E9 and R88 together are responsible for most of the binding affinity of IL-4 for IL4-BP (4). This was evidenced by Zhang et al’s mutational analyses, which showed a 210-fold decrease in affinity for a variant of Y183, and a 2000-fold decrease in affinity for receptor D72 variants. Mutations in peripheral residues caused much smaller decreases in affinity.
Unlike Clusters I and II, Cluster III does not contribute to the affinity of the IL-4/IL4-BP complex, because it cannot form strong hydrogen bonds with the receptor. This is because the geometry of the residues prevents standard sp2-type donor-acceptor interactions. The weaker electrostatic interactions do, however, serve to accelerate complex formation by steering IL-4 into position with its receptor (2).
IL-4 in complex with IL4-BP is similar in structure to free IL-4. However, there is a slight conformational change, involving only a minor shift in the relative orientation of helices. This conformational change stabilizes the overall structure and may also aid in the recruitment of γc to the complex (2) (image 1). IL-4 residues I11, N15, and Y124 are the main residues that bind γc. These residues form a linear epitope besides the IL-4/IL4-BP interface (2). Since IL4-BP can also bind to IL-13, it can be expected that IL-13 and IL-4 will share some structural features at the binding interface. As image 2 shows, positions 121, 124, and 125 of IL-4 overlap closely with corresponding positions on IL-13 (3).
Homologs of the human IL-4/IL4-BP complex were searched using BLAST and DALI. DALI compares the secondary and tertiary structure of the protein with that of other proteins. DALI uses Z-score to measure homology, and a Z-score above 2 indicates significant homology. Interleukin-15 was the most homologous protein, with a Z-score of 10.9 (7). BLAST uses E-value to measure homology, an E-value of 0 indicating perfect homology. BLAST revealed the most homologous protein in terms of primary structure to be the IL-4 isoform 1, found in gorillas, with an E-value of 7e-58 (5).
The IL-4/IL4-BP complex provides a promising area of research for developing drugs that combat asthma. Studying the binding interface between the IL-4 and IL4-BP will give some insight into how other members of the cytokine receptor family that use γc (IL-2, IL-7, IL-9, and IL-15) bind ligands (2). Information on the structure of the interface between IL-4 and IL4-BP will also be constructive towards the development of a drug that mimics IL-4 well enough to block the receptor α chain (2). One application that is already under development is cytokine therapy. Since cytokines dimerize their receptors, with binding of the second chain triggering a sequence of events, mutational analyses can be done to change the affinity of IL-4 for the second chain, γc or IL-13Rα1 (3). The affinity of the complex for γc and IL-13Rα1 is much weaker than the affinity between IL-4 and its binding protein, so the binding site in the IL-4/receptor for γc or IL-13Rα1 might be an easier target for therapy-related drugs than IL-4 to its receptor. This would regulate expression levels of IL-4/IL4BP complex (3). By mutating IL-4 and thus its affinity for a specific type of chain, IL-4 and other cytokines can be engineered to favor binding to certain types of cells, increasing or decreasing the targeted physiological response.