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A cytokine-cytokine interaction in the assembly of higher-order structure and activation of the interleukine-3:receptor complex.

Dey R, Ji K, Liu Z, Chen L - PLoS ONE (2009)

Bottom Line: These observations are consistent with structure-function studies of the GM-CSF:receptor complex showing that formation of the higher-order cytokine:receptor complex is required for signaling.However, a key question not answered from previous studies is how cytokine binding facilitates the assembly of the higher-order complex.Our studies here reveal a potential cytokine-cytokine interaction that participates in the assembly of the dodecamer complex, thus linking cytokine binding to receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Molecular and Computational Biology, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Interleukine-3 (IL-3) binds its receptor and initiates a cascade of signaling processes that regulate the proliferation and differentiation of hematopoietic cells. To understand the detailed mechanisms of IL-3 induced receptor activation, we generated a homology model of the IL-3:receptor complex based on the closely related crystal structure of the GM-CSF:receptor complex. Model-predicted interactions between IL-3 and its receptor are in excellent agreement with mutagenesis data, which validate the model and establish a detailed view of IL-3:receptor interaction. The homology structure reveals an IL-3:IL-3 interaction interface in a higher-order complex modeled after the dodecamer of the GM-CSF:receptor complex wherein an analogous GM-CSF:GM-CSF interface is also identified. This interface is mediated by a proline-rich hydrophobic motif (PPLPLL) of the AA' loop that is highly exposed in the structure of isolated IL-3. Various experimental data suggest that this motif is required for IL-3 function through receptor-binding independent mechanisms. These observations are consistent with structure-function studies of the GM-CSF:receptor complex showing that formation of the higher-order cytokine:receptor complex is required for signaling. However, a key question not answered from previous studies is how cytokine binding facilitates the assembly of the higher-order complex. Our studies here reveal a potential cytokine-cytokine interaction that participates in the assembly of the dodecamer complex, thus linking cytokine binding to receptor activation.

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A cytokine–cytokine interaction interface in the dodecamer.(A) A ribbon/surface presentation of interface IV and V (dashed boxes) in the modeled IL-3:receptor complex. Only domain 4 of the interaction beta subunit and IL-3 are shown in this view. The two residues at interface IV (Glu359 and Lys362) that engage in potential charge repulsion are indicated. The arrows indicate the two fold symmetry axis that runs through both interface IV and V. (B) A top view of interface V in the crystal structure of the GM-CSF:receptor complex (dashed box) and its relative position with respect to other components of the complex. The receptor alpha subunit domain 1 showed only partial density of a few beta strands that are represented by backbone trace here. (C) A top view of interface V (dashed box) mediated by the AA′ loop in the modeled IL-3:receptor complex.
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pone-0005188-g003: A cytokine–cytokine interaction interface in the dodecamer.(A) A ribbon/surface presentation of interface IV and V (dashed boxes) in the modeled IL-3:receptor complex. Only domain 4 of the interaction beta subunit and IL-3 are shown in this view. The two residues at interface IV (Glu359 and Lys362) that engage in potential charge repulsion are indicated. The arrows indicate the two fold symmetry axis that runs through both interface IV and V. (B) A top view of interface V in the crystal structure of the GM-CSF:receptor complex (dashed box) and its relative position with respect to other components of the complex. The receptor alpha subunit domain 1 showed only partial density of a few beta strands that are represented by backbone trace here. (C) A top view of interface V (dashed box) mediated by the AA′ loop in the modeled IL-3:receptor complex.

Mentions: We applied symmetry operations derived from the GM-CSF:receptor crystal to generate both hexamer and dodecamer of the hIL-3:receptor complex (Figure 2A and 2B). The resulting higher-order complex shows that the components of the dodecamer fit nicely with each other. A unique feature of the receptors for GM-CSF, IL-3 and IL-5 is that the beta subunit forms an intertwined dimer [39]. In vitro binding studies suggest that domain 1 and domain 4 from different beta subunits act together to bind the cytokine ligand [40], [41]. The crystal structure of the GM-CSF:receptor complex reveals that this is indeed the case. Domain 1 of one beta subunit and domain 4 of the other form a composite surface to bind GM-CSF at interface II, such that two cytokines and two GMRα chains are cross linked by the beta dimer so as to form a 2∶2∶2 hexamer (Figure 1 in Hansen et al.[34]). In the crystal of the GM-CSF:receptor complex, two hexamers pack head-to-head together to form a dodecamer wherein domain 4 of two adjacent beta subunits contact each other through an extensive protein interface (Figure 3 in Hansen et al. [34]). This interface, referred to as interface IV, has been shown by mutagenesis and neutralizing antibodies to be important for receptor activation. The same interface is also present in the modeled IL-3:receptor complex (Figure 3A). As discussed below, we identified another protein-protein interaction interface involved in the assembly of the GM-CSF:receptor dodecamer (Figure 3B). This interface, termed interface V, is mediated by the loop region between helix A and helix B of GM-CSF bound to two different hexamers. This region of GM-CSF contains a short alpha helix that forms the major part of the interface. In the modeled hIL-3:receptor complex, the corresponding region of IL-3, the AA′ loop, is similarly positioned to form an analogous interaction interface in the dodecamer (Figure 3A and 3C).


A cytokine-cytokine interaction in the assembly of higher-order structure and activation of the interleukine-3:receptor complex.

Dey R, Ji K, Liu Z, Chen L - PLoS ONE (2009)

A cytokine–cytokine interaction interface in the dodecamer.(A) A ribbon/surface presentation of interface IV and V (dashed boxes) in the modeled IL-3:receptor complex. Only domain 4 of the interaction beta subunit and IL-3 are shown in this view. The two residues at interface IV (Glu359 and Lys362) that engage in potential charge repulsion are indicated. The arrows indicate the two fold symmetry axis that runs through both interface IV and V. (B) A top view of interface V in the crystal structure of the GM-CSF:receptor complex (dashed box) and its relative position with respect to other components of the complex. The receptor alpha subunit domain 1 showed only partial density of a few beta strands that are represented by backbone trace here. (C) A top view of interface V (dashed box) mediated by the AA′ loop in the modeled IL-3:receptor complex.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2662821&req=5

pone-0005188-g003: A cytokine–cytokine interaction interface in the dodecamer.(A) A ribbon/surface presentation of interface IV and V (dashed boxes) in the modeled IL-3:receptor complex. Only domain 4 of the interaction beta subunit and IL-3 are shown in this view. The two residues at interface IV (Glu359 and Lys362) that engage in potential charge repulsion are indicated. The arrows indicate the two fold symmetry axis that runs through both interface IV and V. (B) A top view of interface V in the crystal structure of the GM-CSF:receptor complex (dashed box) and its relative position with respect to other components of the complex. The receptor alpha subunit domain 1 showed only partial density of a few beta strands that are represented by backbone trace here. (C) A top view of interface V (dashed box) mediated by the AA′ loop in the modeled IL-3:receptor complex.
Mentions: We applied symmetry operations derived from the GM-CSF:receptor crystal to generate both hexamer and dodecamer of the hIL-3:receptor complex (Figure 2A and 2B). The resulting higher-order complex shows that the components of the dodecamer fit nicely with each other. A unique feature of the receptors for GM-CSF, IL-3 and IL-5 is that the beta subunit forms an intertwined dimer [39]. In vitro binding studies suggest that domain 1 and domain 4 from different beta subunits act together to bind the cytokine ligand [40], [41]. The crystal structure of the GM-CSF:receptor complex reveals that this is indeed the case. Domain 1 of one beta subunit and domain 4 of the other form a composite surface to bind GM-CSF at interface II, such that two cytokines and two GMRα chains are cross linked by the beta dimer so as to form a 2∶2∶2 hexamer (Figure 1 in Hansen et al.[34]). In the crystal of the GM-CSF:receptor complex, two hexamers pack head-to-head together to form a dodecamer wherein domain 4 of two adjacent beta subunits contact each other through an extensive protein interface (Figure 3 in Hansen et al. [34]). This interface, referred to as interface IV, has been shown by mutagenesis and neutralizing antibodies to be important for receptor activation. The same interface is also present in the modeled IL-3:receptor complex (Figure 3A). As discussed below, we identified another protein-protein interaction interface involved in the assembly of the GM-CSF:receptor dodecamer (Figure 3B). This interface, termed interface V, is mediated by the loop region between helix A and helix B of GM-CSF bound to two different hexamers. This region of GM-CSF contains a short alpha helix that forms the major part of the interface. In the modeled hIL-3:receptor complex, the corresponding region of IL-3, the AA′ loop, is similarly positioned to form an analogous interaction interface in the dodecamer (Figure 3A and 3C).

Bottom Line: These observations are consistent with structure-function studies of the GM-CSF:receptor complex showing that formation of the higher-order cytokine:receptor complex is required for signaling.However, a key question not answered from previous studies is how cytokine binding facilitates the assembly of the higher-order complex.Our studies here reveal a potential cytokine-cytokine interaction that participates in the assembly of the dodecamer complex, thus linking cytokine binding to receptor activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Molecular and Computational Biology, University of Southern California, Los Angeles, California, United States of America.

ABSTRACT
Interleukine-3 (IL-3) binds its receptor and initiates a cascade of signaling processes that regulate the proliferation and differentiation of hematopoietic cells. To understand the detailed mechanisms of IL-3 induced receptor activation, we generated a homology model of the IL-3:receptor complex based on the closely related crystal structure of the GM-CSF:receptor complex. Model-predicted interactions between IL-3 and its receptor are in excellent agreement with mutagenesis data, which validate the model and establish a detailed view of IL-3:receptor interaction. The homology structure reveals an IL-3:IL-3 interaction interface in a higher-order complex modeled after the dodecamer of the GM-CSF:receptor complex wherein an analogous GM-CSF:GM-CSF interface is also identified. This interface is mediated by a proline-rich hydrophobic motif (PPLPLL) of the AA' loop that is highly exposed in the structure of isolated IL-3. Various experimental data suggest that this motif is required for IL-3 function through receptor-binding independent mechanisms. These observations are consistent with structure-function studies of the GM-CSF:receptor complex showing that formation of the higher-order cytokine:receptor complex is required for signaling. However, a key question not answered from previous studies is how cytokine binding facilitates the assembly of the higher-order complex. Our studies here reveal a potential cytokine-cytokine interaction that participates in the assembly of the dodecamer complex, thus linking cytokine binding to receptor activation.

Show MeSH