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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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Modeling of the IL-3:receptor complex.(A) The structure of IL-3 (red) is superimposed on that of GM-CSF (yellow). The IL-3 receptor alpha chain domain 2 (α2, blue) is superimposed on the corresponding domain of GM-CSF receptor alpha chain (light magenta). The position of IL-3 receptor alpha chain domain 1 (α1, blue) is fitted as described in text. Domain 1 of one beta subunit (β1) and domain 4 of the other (β4′) are shown in green. The rest of the beta dimer is not shown. The three interfaces of the complex discussed in the text are labeled. The color scheme is used throughout the illustration unless otherwise indicated; (B) The resulting model of the IL-3:receptor complex after removing GM-CSF and its receptor alpha subunit, shown in ribbon covered with transparent surface; (c)sequence alignment of IL-3, GM-CSF and IL-5. Helices in IL-3 are indicated by horizontal bar in red. Residues discussed in the text are highlighted in different colors: green shade, residues interacting with the receptor β-subunit in IL-3 and GM-CSF; blue shade, residues of IL-3 that interact with the receptor α-subunit; magenta shade, residues of GM-CSF that interact with the receptor α-subunit; red font, functionally important residues from mutagenesis studies. Boxed residues are involved interface V interaction. Residue numbering above the sequence is for IL-3.
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pone-0005188-g001: Modeling of the IL-3:receptor complex.(A) The structure of IL-3 (red) is superimposed on that of GM-CSF (yellow). The IL-3 receptor alpha chain domain 2 (α2, blue) is superimposed on the corresponding domain of GM-CSF receptor alpha chain (light magenta). The position of IL-3 receptor alpha chain domain 1 (α1, blue) is fitted as described in text. Domain 1 of one beta subunit (β1) and domain 4 of the other (β4′) are shown in green. The rest of the beta dimer is not shown. The three interfaces of the complex discussed in the text are labeled. The color scheme is used throughout the illustration unless otherwise indicated; (B) The resulting model of the IL-3:receptor complex after removing GM-CSF and its receptor alpha subunit, shown in ribbon covered with transparent surface; (c)sequence alignment of IL-3, GM-CSF and IL-5. Helices in IL-3 are indicated by horizontal bar in red. Residues discussed in the text are highlighted in different colors: green shade, residues interacting with the receptor β-subunit in IL-3 and GM-CSF; blue shade, residues of IL-3 that interact with the receptor α-subunit; magenta shade, residues of GM-CSF that interact with the receptor α-subunit; red font, functionally important residues from mutagenesis studies. Boxed residues are involved interface V interaction. Residue numbering above the sequence is for IL-3.

Mentions: The structure of isolated GM-CSF is similar to its receptor bound form [34], [38]. We assume that the structure of hIL-3 bound to its receptor is also similar to its free from. We therefore docked IL-3 as a rigid body on the GM-CSF:receptor complex by superimposing its structure with that of GM-CSF (Figure 1A). The superposition is based on the backbone of the four conserved helices (A–D) that shows an RMSD of 2.7 Å for 50 C-α atoms (Figure S4). The homology model of hIL-3 receptor α-chain is docked onto the GM-CSF:receptor complex by superimposing its domain 2 with that of GM-CSF receptor α-chains (Figure S5A). Domain 1 of hIL-3 receptor α-chain, which is constructed based on the template of IL-13RA domain1 (Figure S5), is fitted into the ternary complex based on the inter-domain orientation of IL-13RA with minor manual adjustments (see below). GM-CSF and its receptor alpha chain were then removed from the combined coordinates. With the remaining common β-chain, hIL-3 and the receptor alpha chain were therefore placed together using the molecular framework defined by the crystal structure of the GM-CSF:receptor complex (Figure 1B). The complex model was subject to energy minimization with fixed backbone using MODELLER. A few local clashes in the final model were removed manually in O by choosing different side chain rotamers. Throughout the modeling process, only domain 1 and domain 4 from two different hIL-3 receptor β-subunits that are in contact with IL-3 were included in the model refinement procedures. A multiple sequence alignment among IL-3, GM-CSF, and IL-5 is shown in Figure 1C with residues discussed in the text highlighted. Overall, the homology model is based on the identical beta subunit and closely related cytokine structures between IL-3 and GM-CSF. Although the accuracy of the detailed hIL-3 receptor α-chain structure may be limited by the modest sequence identity, the overall arrangement of its two domains in the final complex can be assigned with high confidence, so the role of the receptor α-chain in the assembly of the dodecamer complex can be assessed with reasonable certainty (see below).


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)

Modeling of the IL-3:receptor complex.(A) The structure of IL-3 (red) is superimposed on that of GM-CSF (yellow). The IL-3 receptor alpha chain domain 2 (α2, blue) is superimposed on the corresponding domain of GM-CSF receptor alpha chain (light magenta). The position of IL-3 receptor alpha chain domain 1 (α1, blue) is fitted as described in text. Domain 1 of one beta subunit (β1) and domain 4 of the other (β4′) are shown in green. The rest of the beta dimer is not shown. The three interfaces of the complex discussed in the text are labeled. The color scheme is used throughout the illustration unless otherwise indicated; (B) The resulting model of the IL-3:receptor complex after removing GM-CSF and its receptor alpha subunit, shown in ribbon covered with transparent surface; (c)sequence alignment of IL-3, GM-CSF and IL-5. Helices in IL-3 are indicated by horizontal bar in red. Residues discussed in the text are highlighted in different colors: green shade, residues interacting with the receptor β-subunit in IL-3 and GM-CSF; blue shade, residues of IL-3 that interact with the receptor α-subunit; magenta shade, residues of GM-CSF that interact with the receptor α-subunit; red font, functionally important residues from mutagenesis studies. Boxed residues are involved interface V interaction. Residue numbering above the sequence is for IL-3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662821&req=5

pone-0005188-g001: Modeling of the IL-3:receptor complex.(A) The structure of IL-3 (red) is superimposed on that of GM-CSF (yellow). The IL-3 receptor alpha chain domain 2 (α2, blue) is superimposed on the corresponding domain of GM-CSF receptor alpha chain (light magenta). The position of IL-3 receptor alpha chain domain 1 (α1, blue) is fitted as described in text. Domain 1 of one beta subunit (β1) and domain 4 of the other (β4′) are shown in green. The rest of the beta dimer is not shown. The three interfaces of the complex discussed in the text are labeled. The color scheme is used throughout the illustration unless otherwise indicated; (B) The resulting model of the IL-3:receptor complex after removing GM-CSF and its receptor alpha subunit, shown in ribbon covered with transparent surface; (c)sequence alignment of IL-3, GM-CSF and IL-5. Helices in IL-3 are indicated by horizontal bar in red. Residues discussed in the text are highlighted in different colors: green shade, residues interacting with the receptor β-subunit in IL-3 and GM-CSF; blue shade, residues of IL-3 that interact with the receptor α-subunit; magenta shade, residues of GM-CSF that interact with the receptor α-subunit; red font, functionally important residues from mutagenesis studies. Boxed residues are involved interface V interaction. Residue numbering above the sequence is for IL-3.
Mentions: The structure of isolated GM-CSF is similar to its receptor bound form [34], [38]. We assume that the structure of hIL-3 bound to its receptor is also similar to its free from. We therefore docked IL-3 as a rigid body on the GM-CSF:receptor complex by superimposing its structure with that of GM-CSF (Figure 1A). The superposition is based on the backbone of the four conserved helices (A–D) that shows an RMSD of 2.7 Å for 50 C-α atoms (Figure S4). The homology model of hIL-3 receptor α-chain is docked onto the GM-CSF:receptor complex by superimposing its domain 2 with that of GM-CSF receptor α-chains (Figure S5A). Domain 1 of hIL-3 receptor α-chain, which is constructed based on the template of IL-13RA domain1 (Figure S5), is fitted into the ternary complex based on the inter-domain orientation of IL-13RA with minor manual adjustments (see below). GM-CSF and its receptor alpha chain were then removed from the combined coordinates. With the remaining common β-chain, hIL-3 and the receptor alpha chain were therefore placed together using the molecular framework defined by the crystal structure of the GM-CSF:receptor complex (Figure 1B). The complex model was subject to energy minimization with fixed backbone using MODELLER. A few local clashes in the final model were removed manually in O by choosing different side chain rotamers. Throughout the modeling process, only domain 1 and domain 4 from two different hIL-3 receptor β-subunits that are in contact with IL-3 were included in the model refinement procedures. A multiple sequence alignment among IL-3, GM-CSF, and IL-5 is shown in Figure 1C with residues discussed in the text highlighted. Overall, the homology model is based on the identical beta subunit and closely related cytokine structures between IL-3 and GM-CSF. Although the accuracy of the detailed hIL-3 receptor α-chain structure may be limited by the modest sequence identity, the overall arrangement of its two domains in the final complex can be assigned with high confidence, so the role of the receptor α-chain in the assembly of the dodecamer complex can be assessed with reasonable certainty (see below).

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