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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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Mutations to the allosteric network producing biochemical phenotypes.(a) D404N and D386N mutations leading to stronger substrate binding and disrupting the binding cooperativity, respectively. (b) Effect of W260A on ATP-binding affinity. (c) Effect of W260A on peptide-binding affinity. (d) Effect of T338I on ATP-binding affinity. (e) Effect of T338I on substrate-binding affinity. Kd values were determined using fluorescence anisotropy at 1 μM labelled Src-optimized peptide (Rs1). All experiments were performed in triplicate and data represent mean values±s.e.m.
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f4: Mutations to the allosteric network producing biochemical phenotypes.(a) D404N and D386N mutations leading to stronger substrate binding and disrupting the binding cooperativity, respectively. (b) Effect of W260A on ATP-binding affinity. (c) Effect of W260A on peptide-binding affinity. (d) Effect of T338I on ATP-binding affinity. (e) Effect of T338I on substrate-binding affinity. Kd values were determined using fluorescence anisotropy at 1 μM labelled Src-optimized peptide (Rs1). All experiments were performed in triplicate and data represent mean values±s.e.m.

Mentions: Our simulations suggest that the protonation of Asp404 of the DFG motif may switch the allosteric network from one conformational state to another. Moreover, our experiments show that ATP binding weakens the substrate binding, while, on the other hand, ADP binding strengthens substrate-peptide binding (Fig. 3d). Since ATP and ADP binding are associated with a deprotonated and protonated DFG aspartate, respectively, we infer that the conformational state that is favoured by DFG protonation is more capable of substrate binding. To examine this possibility, we replaced Asp404 of the DFG motif with asparagine to mimic the protonation of the residue39 (Supplementary Fig. 10). We found, as anticipated, that Src D404N binds peptides with 12-fold higher affinity than the wild type (Fig. 4a). The asparagine residue is incapable of chelating Mg2+ ions and it thus does not bind ATP favourably (Supplementary Fig. 10c). These findings regarding the D404N mutant further support both the role of the DFG motif as a central conformational switch14 and the residue-specific structural mechanism associated with the allosteric network we propose.


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

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

Mutations to the allosteric network producing biochemical phenotypes.(a) D404N and D386N mutations leading to stronger substrate binding and disrupting the binding cooperativity, respectively. (b) Effect of W260A on ATP-binding affinity. (c) Effect of W260A on peptide-binding affinity. (d) Effect of T338I on ATP-binding affinity. (e) Effect of T338I on substrate-binding affinity. Kd values were determined using fluorescence anisotropy at 1 μM labelled Src-optimized peptide (Rs1). 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

f4: Mutations to the allosteric network producing biochemical phenotypes.(a) D404N and D386N mutations leading to stronger substrate binding and disrupting the binding cooperativity, respectively. (b) Effect of W260A on ATP-binding affinity. (c) Effect of W260A on peptide-binding affinity. (d) Effect of T338I on ATP-binding affinity. (e) Effect of T338I on substrate-binding affinity. Kd values were determined using fluorescence anisotropy at 1 μM labelled Src-optimized peptide (Rs1). All experiments were performed in triplicate and data represent mean values±s.e.m.
Mentions: Our simulations suggest that the protonation of Asp404 of the DFG motif may switch the allosteric network from one conformational state to another. Moreover, our experiments show that ATP binding weakens the substrate binding, while, on the other hand, ADP binding strengthens substrate-peptide binding (Fig. 3d). Since ATP and ADP binding are associated with a deprotonated and protonated DFG aspartate, respectively, we infer that the conformational state that is favoured by DFG protonation is more capable of substrate binding. To examine this possibility, we replaced Asp404 of the DFG motif with asparagine to mimic the protonation of the residue39 (Supplementary Fig. 10). We found, as anticipated, that Src D404N binds peptides with 12-fold higher affinity than the wild type (Fig. 4a). The asparagine residue is incapable of chelating Mg2+ ions and it thus does not bind ATP favourably (Supplementary Fig. 10c). These findings regarding the D404N mutant further support both the role of the DFG motif as a central conformational switch14 and the residue-specific structural mechanism associated with the allosteric network we propose.

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