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A dynamically coupled allosteric network underlies binding cooperativity in Src kinase.

Foda ZH, Shan Y, Kim ET, Shaw DE, Seeliger MA - Nat Commun (2015)

Bottom Line: Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity.We confirm the molecular details of the signal relay through the allosteric network by biochemical studies.Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794, USA.

ABSTRACT
Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

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Negative cooperativity of ATP and substrate binding and positive cooperativity of ADP and substrate binding.(a) The effect of ATP concentration on substrate Km for Src, Abl and Hck kinase domains. (b) The effect of substrate peptide concentration on ATP Km for Src, Abl and Hck kinase domains. (c) The effect of AMP-PNP concentration on substrate Kd for Src, Abl and Hck kinase domains. (d) The effect of ADP and AMP-PNP concentration on substrate Kd for the Src kinase domain. (e) Dissociation constants for peptides of different sequences at increasing AMP-PNP concentration. Rs1: Src-optimal substrate peptide (AEEEIYGEFAKKK); Rs2: peptide sequence (AEEMIYGEFAKKK); Rs3: peptide sequence (GIYWHHY). Km values were determined in a kinase activity assay. Kd values for the substrate peptides were determined using fluorescence anisotropy at 1 μM labelled peptide. Kd values for AMP-PNP were determined using isothermal titration calorimetry. (f) The effect of peptide and phosphorylated peptide on ADP and AMP-PNP binding. All experiments were performed in triplicate and data represent mean values±s.e.m.
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f3: Negative cooperativity of ATP and substrate binding and positive cooperativity of ADP and substrate binding.(a) The effect of ATP concentration on substrate Km for Src, Abl and Hck kinase domains. (b) The effect of substrate peptide concentration on ATP Km for Src, Abl and Hck kinase domains. (c) The effect of AMP-PNP concentration on substrate Kd for Src, Abl and Hck kinase domains. (d) The effect of ADP and AMP-PNP concentration on substrate Kd for the Src kinase domain. (e) Dissociation constants for peptides of different sequences at increasing AMP-PNP concentration. Rs1: Src-optimal substrate peptide (AEEEIYGEFAKKK); Rs2: peptide sequence (AEEMIYGEFAKKK); Rs3: peptide sequence (GIYWHHY). Km values were determined in a kinase activity assay. Kd values for the substrate peptides were determined using fluorescence anisotropy at 1 μM labelled peptide. Kd values for AMP-PNP were determined using isothermal titration calorimetry. (f) The effect of peptide and phosphorylated peptide on ADP and AMP-PNP binding. All experiments were performed in triplicate and data represent mean values±s.e.m.

Mentions: The putative allosteric network discussed above may manifest itself in the form of cooperativity between ATP and substrate binding. While positive cooperativity between ATP and substrate binding has been established for the Ser/Thr kinase PKA33, such cooperativity has not previously been observed in a tyrosine kinase. Using a fluorescently labelled optimal substrate peptide for Src34, we determined a dissociation constant (Kd) of 37±7 (s.e.m.) μM for the interaction of the Src kinase domain with the peptide in the absence of ATP. Higher concentrations of AMP-PNP, a non-hydrolysable ATP analogue, increase the Kd up to fivefold (Fig. 3c), which indicates negative binding cooperativity. To study the effect of this negative binding cooperativity on kinase activity, we conducted kinase activity assays. We determined the Michaelis–Menten constant (Km) for substrate peptide and ATP at varying ATP and peptide concentrations, respectively. We found that the Km for peptide increases threefold over an ATP concentration range from 50 to 800 μM (Fig. 3a), while the Km for ATP increases from 54±22 μM at 50 μM substrate peptide to 202±21 μM in the presence of 800 μM substrate peptide (Fig. 3b).


A dynamically coupled allosteric network underlies binding cooperativity in Src kinase.

Foda ZH, Shan Y, Kim ET, Shaw DE, Seeliger MA - Nat Commun (2015)

Negative cooperativity of ATP and substrate binding and positive cooperativity of ADP and substrate binding.(a) The effect of ATP concentration on substrate Km for Src, Abl and Hck kinase domains. (b) The effect of substrate peptide concentration on ATP Km for Src, Abl and Hck kinase domains. (c) The effect of AMP-PNP concentration on substrate Kd for Src, Abl and Hck kinase domains. (d) The effect of ADP and AMP-PNP concentration on substrate Kd for the Src kinase domain. (e) Dissociation constants for peptides of different sequences at increasing AMP-PNP concentration. Rs1: Src-optimal substrate peptide (AEEEIYGEFAKKK); Rs2: peptide sequence (AEEMIYGEFAKKK); Rs3: peptide sequence (GIYWHHY). Km values were determined in a kinase activity assay. Kd values for the substrate peptides were determined using fluorescence anisotropy at 1 μM labelled peptide. Kd values for AMP-PNP were determined using isothermal titration calorimetry. (f) The effect of peptide and phosphorylated peptide on ADP and AMP-PNP binding. All experiments were performed in triplicate and data represent mean values±s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300553&req=5

f3: Negative cooperativity of ATP and substrate binding and positive cooperativity of ADP and substrate binding.(a) The effect of ATP concentration on substrate Km for Src, Abl and Hck kinase domains. (b) The effect of substrate peptide concentration on ATP Km for Src, Abl and Hck kinase domains. (c) The effect of AMP-PNP concentration on substrate Kd for Src, Abl and Hck kinase domains. (d) The effect of ADP and AMP-PNP concentration on substrate Kd for the Src kinase domain. (e) Dissociation constants for peptides of different sequences at increasing AMP-PNP concentration. Rs1: Src-optimal substrate peptide (AEEEIYGEFAKKK); Rs2: peptide sequence (AEEMIYGEFAKKK); Rs3: peptide sequence (GIYWHHY). Km values were determined in a kinase activity assay. Kd values for the substrate peptides were determined using fluorescence anisotropy at 1 μM labelled peptide. Kd values for AMP-PNP were determined using isothermal titration calorimetry. (f) The effect of peptide and phosphorylated peptide on ADP and AMP-PNP binding. All experiments were performed in triplicate and data represent mean values±s.e.m.
Mentions: The putative allosteric network discussed above may manifest itself in the form of cooperativity between ATP and substrate binding. While positive cooperativity between ATP and substrate binding has been established for the Ser/Thr kinase PKA33, such cooperativity has not previously been observed in a tyrosine kinase. Using a fluorescently labelled optimal substrate peptide for Src34, we determined a dissociation constant (Kd) of 37±7 (s.e.m.) μM for the interaction of the Src kinase domain with the peptide in the absence of ATP. Higher concentrations of AMP-PNP, a non-hydrolysable ATP analogue, increase the Kd up to fivefold (Fig. 3c), which indicates negative binding cooperativity. To study the effect of this negative binding cooperativity on kinase activity, we conducted kinase activity assays. We determined the Michaelis–Menten constant (Km) for substrate peptide and ATP at varying ATP and peptide concentrations, respectively. We found that the Km for peptide increases threefold over an ATP concentration range from 50 to 800 μM (Fig. 3a), while the Km for ATP increases from 54±22 μM at 50 μM substrate peptide to 202±21 μM in the presence of 800 μM substrate peptide (Fig. 3b).

Bottom Line: Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity.We confirm the molecular details of the signal relay through the allosteric network by biochemical studies.Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794, USA.

ABSTRACT
Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

Show MeSH
Related in: MedlinePlus