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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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The allosteric network and negative cooperativity in the context of a kinase catalytic cycle.(a) The key components of the allosteric network are shown in two configurations. The protonation of the DFG aspartate repositions the Asp and Phe residues, which disrupts the regulatory hydrophobic spine and leads to the αC-out transition and the repositioning of Trp260 in the N-lobe, and in the C-lobe induces the RAA/AAR Arg to form alternative interactions with Trp428 and Glu454, resulting in a rearrangement of the substrate-binding site. (b) In a catalytic cycle, the apo kinase (I) binds ATP/Mg2+ and substrate, yielding the bisubstrate complex (II), in which the phosphoryl transfer from ATP to substrate occurs. Following the phosphoryl-transfer step, the DFG aspartate becomes protonated (III). The phosphorylated substrate is subsequently released (IV), which weakens ADP binding through the cooperative mechanism and promotes ADP release. The DFG aspartate is then once again deprotonated, and the affinity for ATP increases, starting the catalytic cycle again (I).
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f5: The allosteric network and negative cooperativity in the context of a kinase catalytic cycle.(a) The key components of the allosteric network are shown in two configurations. The protonation of the DFG aspartate repositions the Asp and Phe residues, which disrupts the regulatory hydrophobic spine and leads to the αC-out transition and the repositioning of Trp260 in the N-lobe, and in the C-lobe induces the RAA/AAR Arg to form alternative interactions with Trp428 and Glu454, resulting in a rearrangement of the substrate-binding site. (b) In a catalytic cycle, the apo kinase (I) binds ATP/Mg2+ and substrate, yielding the bisubstrate complex (II), in which the phosphoryl transfer from ATP to substrate occurs. Following the phosphoryl-transfer step, the DFG aspartate becomes protonated (III). The phosphorylated substrate is subsequently released (IV), which weakens ADP binding through the cooperative mechanism and promotes ADP release. The DFG aspartate is then once again deprotonated, and the affinity for ATP increases, starting the catalytic cycle again (I).

Mentions: Having investigated how a perturbation at the catalytic site (the protonation of the DFG aspartate as a result of local electrostatic changes) may propagate through the Src kinase domain, we propose a dynamically coupled allosteric network connecting the protein’s ATP- and substrate-binding sites (Fig. 5a). The protonation repositions the tightly coupled aspartate and phenylalanine of the DFG motif. In the N-lobe, the repositioning of the phenylalanine disrupts the regulatory hydrophobic spine and leads to the αC-out transition and the repositioning of Trp260. In the C-lobe, the protonated aspartate shares a hydrogen bond with the aspartate of the conserved HRD motif, leading to displacement of the conserved arginine (Arg388) of the RAA/AAR motif, which in turn forms alternative interactions with Trp428 and Glu454, resulting in a rearrangement of the substrate-binding site.


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

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

The allosteric network and negative cooperativity in the context of a kinase catalytic cycle.(a) The key components of the allosteric network are shown in two configurations. The protonation of the DFG aspartate repositions the Asp and Phe residues, which disrupts the regulatory hydrophobic spine and leads to the αC-out transition and the repositioning of Trp260 in the N-lobe, and in the C-lobe induces the RAA/AAR Arg to form alternative interactions with Trp428 and Glu454, resulting in a rearrangement of the substrate-binding site. (b) In a catalytic cycle, the apo kinase (I) binds ATP/Mg2+ and substrate, yielding the bisubstrate complex (II), in which the phosphoryl transfer from ATP to substrate occurs. Following the phosphoryl-transfer step, the DFG aspartate becomes protonated (III). The phosphorylated substrate is subsequently released (IV), which weakens ADP binding through the cooperative mechanism and promotes ADP release. The DFG aspartate is then once again deprotonated, and the affinity for ATP increases, starting the catalytic cycle again (I).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: The allosteric network and negative cooperativity in the context of a kinase catalytic cycle.(a) The key components of the allosteric network are shown in two configurations. The protonation of the DFG aspartate repositions the Asp and Phe residues, which disrupts the regulatory hydrophobic spine and leads to the αC-out transition and the repositioning of Trp260 in the N-lobe, and in the C-lobe induces the RAA/AAR Arg to form alternative interactions with Trp428 and Glu454, resulting in a rearrangement of the substrate-binding site. (b) In a catalytic cycle, the apo kinase (I) binds ATP/Mg2+ and substrate, yielding the bisubstrate complex (II), in which the phosphoryl transfer from ATP to substrate occurs. Following the phosphoryl-transfer step, the DFG aspartate becomes protonated (III). The phosphorylated substrate is subsequently released (IV), which weakens ADP binding through the cooperative mechanism and promotes ADP release. The DFG aspartate is then once again deprotonated, and the affinity for ATP increases, starting the catalytic cycle again (I).
Mentions: Having investigated how a perturbation at the catalytic site (the protonation of the DFG aspartate as a result of local electrostatic changes) may propagate through the Src kinase domain, we propose a dynamically coupled allosteric network connecting the protein’s ATP- and substrate-binding sites (Fig. 5a). The protonation repositions the tightly coupled aspartate and phenylalanine of the DFG motif. In the N-lobe, the repositioning of the phenylalanine disrupts the regulatory hydrophobic spine and leads to the αC-out transition and the repositioning of Trp260. In the C-lobe, the protonated aspartate shares a hydrogen bond with the aspartate of the conserved HRD motif, leading to displacement of the conserved arginine (Arg388) of the RAA/AAR motif, which in turn forms alternative interactions with Trp428 and Glu454, resulting in a rearrangement of the substrate-binding site.

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