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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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Protein–protein interactions at interfaces I and III.(A) A charged interaction patch between helix A of IL-3 and domain 2 of IL-3R alpha. Here Asp21 and Glu119 of IL-3 engage in electrostatic interaction with Arg277 and Arg234 of IL-3R alpha, respectively. (B) A hydrophobic interaction patch between IL-3 and IL-3R alpha domain 1. Note that Phe202 of IL-3R alpha inserts into a hydrophobic pocket formed by Phe113, Thr112 and the aliphatic side chain of Lys116 on IL-3. (C) Foreground: the loop region between Arg257 and Gln261 of IL-3R alpha (blue) is very similar to the corresponding region of GMR alpha (magenta) in sequence, structure and interaction with beta-subunit (green). Background: residues Lys244, Arg245 and Gln247 of IL-3R alpha also contact the beta subunit.
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pone-0005188-g005: Protein–protein interactions at interfaces I and III.(A) A charged interaction patch between helix A of IL-3 and domain 2 of IL-3R alpha. Here Asp21 and Glu119 of IL-3 engage in electrostatic interaction with Arg277 and Arg234 of IL-3R alpha, respectively. (B) A hydrophobic interaction patch between IL-3 and IL-3R alpha domain 1. Note that Phe202 of IL-3R alpha inserts into a hydrophobic pocket formed by Phe113, Thr112 and the aliphatic side chain of Lys116 on IL-3. (C) Foreground: the loop region between Arg257 and Gln261 of IL-3R alpha (blue) is very similar to the corresponding region of GMR alpha (magenta) in sequence, structure and interaction with beta-subunit (green). Background: residues Lys244, Arg245 and Gln247 of IL-3R alpha also contact the beta subunit.

Mentions: At interface I, helix A, A′ and D of IL-3 bind domain 1 and domain 2 of IL-3R alpha through two distinct patches of protein-protein interactions, one dominated by electrostatic interactions while the other rich in hydrophobic/van der Waals contacts. The charge interaction patch is formed between helix A of IL-3 and domain 2 of IL-3R alpha, where Asp21 and Glu119 of IL-3 are in position to engage in electrostatic interaction with Arg277 and Arg234 of IL-3R alpha, respectively (Figure 5A). Arg234 of IL-3R alpha is also in position to interact with Ser17 of IL-3 through hydrogen bonding. For the hydrophobic patch, residues located on various loops of IL-3R alpha domain 1, including Val200, Phe202, Ser177–Ser179, and Ala143-Arg146, project toward the cytokine to interact with Gln124, Asn120, Thr117, Lys116, Phe113, Gly42, Gln45, Asp46, and Met49 on helix D and A′ of IL-3. Most notably, Phe202 of IL-3R alpha is poised to insert into a hydrophobic pocket formed by Phe113, Thr112 and the aliphatic side chain of Lys116 on IL-3 (Figure 5B). Consistent with the structure model, a number of IL-3 residues located at interface I, including Ser17, Asn18, Asp21, Arg108, Phe113, Lys116 and Glu119 have been shown to be important for receptor binding and activation by mutagenesis [29], [30], [32] (Figure 1C). The loss of function for mutations Ser17Lys, Asn18Lys, Thr25Arg and Glu119Arg of IL-3 could be explained by charge repulsion with Arg234 and Arg277 of IL-3R alpha [32], while that for Phe113A could be explained by diminished hydrophobic interactions with Phe202 of IL-3R alpha. Interestingly, a number of IL-3 mutations, such as Lys116Val, Gln45Val, Lys116Trp, and Thr112Arg, showed enhancement in receptor binding and/or cellular activities [29], [31], [32]. Remarkably, these residues are located around Phe113 in the folded structure of IL-3. Substitution of these hydrophilic residues with hydrophobic residues or residues with longer aliphatic side chain could therefore augment the hydrophobic interaction at interface I and hence enhance the activity of mutated IL-3.


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)

Protein–protein interactions at interfaces I and III.(A) A charged interaction patch between helix A of IL-3 and domain 2 of IL-3R alpha. Here Asp21 and Glu119 of IL-3 engage in electrostatic interaction with Arg277 and Arg234 of IL-3R alpha, respectively. (B) A hydrophobic interaction patch between IL-3 and IL-3R alpha domain 1. Note that Phe202 of IL-3R alpha inserts into a hydrophobic pocket formed by Phe113, Thr112 and the aliphatic side chain of Lys116 on IL-3. (C) Foreground: the loop region between Arg257 and Gln261 of IL-3R alpha (blue) is very similar to the corresponding region of GMR alpha (magenta) in sequence, structure and interaction with beta-subunit (green). Background: residues Lys244, Arg245 and Gln247 of IL-3R alpha also contact the beta subunit.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2662821&req=5

pone-0005188-g005: Protein–protein interactions at interfaces I and III.(A) A charged interaction patch between helix A of IL-3 and domain 2 of IL-3R alpha. Here Asp21 and Glu119 of IL-3 engage in electrostatic interaction with Arg277 and Arg234 of IL-3R alpha, respectively. (B) A hydrophobic interaction patch between IL-3 and IL-3R alpha domain 1. Note that Phe202 of IL-3R alpha inserts into a hydrophobic pocket formed by Phe113, Thr112 and the aliphatic side chain of Lys116 on IL-3. (C) Foreground: the loop region between Arg257 and Gln261 of IL-3R alpha (blue) is very similar to the corresponding region of GMR alpha (magenta) in sequence, structure and interaction with beta-subunit (green). Background: residues Lys244, Arg245 and Gln247 of IL-3R alpha also contact the beta subunit.
Mentions: At interface I, helix A, A′ and D of IL-3 bind domain 1 and domain 2 of IL-3R alpha through two distinct patches of protein-protein interactions, one dominated by electrostatic interactions while the other rich in hydrophobic/van der Waals contacts. The charge interaction patch is formed between helix A of IL-3 and domain 2 of IL-3R alpha, where Asp21 and Glu119 of IL-3 are in position to engage in electrostatic interaction with Arg277 and Arg234 of IL-3R alpha, respectively (Figure 5A). Arg234 of IL-3R alpha is also in position to interact with Ser17 of IL-3 through hydrogen bonding. For the hydrophobic patch, residues located on various loops of IL-3R alpha domain 1, including Val200, Phe202, Ser177–Ser179, and Ala143-Arg146, project toward the cytokine to interact with Gln124, Asn120, Thr117, Lys116, Phe113, Gly42, Gln45, Asp46, and Met49 on helix D and A′ of IL-3. Most notably, Phe202 of IL-3R alpha is poised to insert into a hydrophobic pocket formed by Phe113, Thr112 and the aliphatic side chain of Lys116 on IL-3 (Figure 5B). Consistent with the structure model, a number of IL-3 residues located at interface I, including Ser17, Asn18, Asp21, Arg108, Phe113, Lys116 and Glu119 have been shown to be important for receptor binding and activation by mutagenesis [29], [30], [32] (Figure 1C). The loss of function for mutations Ser17Lys, Asn18Lys, Thr25Arg and Glu119Arg of IL-3 could be explained by charge repulsion with Arg234 and Arg277 of IL-3R alpha [32], while that for Phe113A could be explained by diminished hydrophobic interactions with Phe202 of IL-3R alpha. Interestingly, a number of IL-3 mutations, such as Lys116Val, Gln45Val, Lys116Trp, and Thr112Arg, showed enhancement in receptor binding and/or cellular activities [29], [31], [32]. Remarkably, these residues are located around Phe113 in the folded structure of IL-3. Substitution of these hydrophilic residues with hydrophobic residues or residues with longer aliphatic side chain could therefore augment the hydrophobic interaction at interface I and hence enhance the activity of mutated IL-3.

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