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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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Atomic details of the concerted conformational change.(a) An overview of the active Src kinase domain (from PDB entry 1Y57). Helix αC is coloured orange, P-loop red, activation loop blue, catalytic loop pink, P+1 loop cyan and helix αG with αG-αF loop green. Black rectangle indicates the enlarged area to the right. (b,c) Close-up of the residues identified as part of the allosteric network before (b) and after (c) the transition is observed in the simulations. The P-loop and parts of the activation loop have been omitted for clarity. The hydrophobic spine residues (orange transparent surface) and the substrate-binding site (green transparent surface) are highlighted. (b) Prepared using PDB 1Y57, from which the simulation was initiated. (c) Prepared using a snapshot from a simulation, but the key features highlighted (for example, the positions of Trp260 and the Glu310-Arg409 salt bridge) are also seen in the crystal structure of inactive Src kinase (PDB 2SRC). The key hydrogen bond between protonated Asp404 and Asp386 is seen in other kinase crystal structures (for example, PDB 1JOW and 1M14).
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f2: Atomic details of the concerted conformational change.(a) An overview of the active Src kinase domain (from PDB entry 1Y57). Helix αC is coloured orange, P-loop red, activation loop blue, catalytic loop pink, P+1 loop cyan and helix αG with αG-αF loop green. Black rectangle indicates the enlarged area to the right. (b,c) Close-up of the residues identified as part of the allosteric network before (b) and after (c) the transition is observed in the simulations. The P-loop and parts of the activation loop have been omitted for clarity. The hydrophobic spine residues (orange transparent surface) and the substrate-binding site (green transparent surface) are highlighted. (b) Prepared using PDB 1Y57, from which the simulation was initiated. (c) Prepared using a snapshot from a simulation, but the key features highlighted (for example, the positions of Trp260 and the Glu310-Arg409 salt bridge) are also seen in the crystal structure of inactive Src kinase (PDB 2SRC). The key hydrogen bond between protonated Asp404 and Asp386 is seen in other kinase crystal structures (for example, PDB 1JOW and 1M14).

Mentions: A conserved hydrophobic ‘spine’ in the protein kinase domain plays a critical role in maintaining the catalytically active conformation20. Consistent with this notion, the αC-out transition was accompanied in our simulations by the breaking of the hydrophobic spine (Leu325, Met314 and Phe405 of the DFG motif, and His384 in Src kinase) in the N-lobe (Figs 1c and 2b). Presumably, the protonation of Asp404 of the DFG motif leads the residue to disengage from Lys295; by the coupling of the DFG residues, this conformational change at Asp404 causes Phe405 to disengage from Met314, breaking the spine and repositioning the αC helix (Fig. 2b). We also observed that Trp260, the first residue of the kinase domain and a key residue in the cross-talk between the regulatory SH2/SH3 and kinase domains in Src2122, interacts with Leu325 and thus the hydrophobic spine (Fig. 2b). During the αC-out transition observed in the simulations, Trp260 changed position (Fig. 2b). Given that this repositioning of Trp260 is consistent with the different Trp260 conformations in active and inactive Src kinase structures (this conformation of Trp260 after the αC-out transition has been observed in X-ray structures of inactive Src kinase2324) and that it coincides with the αC-out transition in both of the two separate simulations in which the transition is observed (Fig. 1c, Plot A and Supplementary Fig. 2a, Plot A), we conclude that Trp260 is likely conformationally coupled with the hydrophobic spine, and the αC helix.


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

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

Atomic details of the concerted conformational change.(a) An overview of the active Src kinase domain (from PDB entry 1Y57). Helix αC is coloured orange, P-loop red, activation loop blue, catalytic loop pink, P+1 loop cyan and helix αG with αG-αF loop green. Black rectangle indicates the enlarged area to the right. (b,c) Close-up of the residues identified as part of the allosteric network before (b) and after (c) the transition is observed in the simulations. The P-loop and parts of the activation loop have been omitted for clarity. The hydrophobic spine residues (orange transparent surface) and the substrate-binding site (green transparent surface) are highlighted. (b) Prepared using PDB 1Y57, from which the simulation was initiated. (c) Prepared using a snapshot from a simulation, but the key features highlighted (for example, the positions of Trp260 and the Glu310-Arg409 salt bridge) are also seen in the crystal structure of inactive Src kinase (PDB 2SRC). The key hydrogen bond between protonated Asp404 and Asp386 is seen in other kinase crystal structures (for example, PDB 1JOW and 1M14).
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Related In: Results  -  Collection

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

f2: Atomic details of the concerted conformational change.(a) An overview of the active Src kinase domain (from PDB entry 1Y57). Helix αC is coloured orange, P-loop red, activation loop blue, catalytic loop pink, P+1 loop cyan and helix αG with αG-αF loop green. Black rectangle indicates the enlarged area to the right. (b,c) Close-up of the residues identified as part of the allosteric network before (b) and after (c) the transition is observed in the simulations. The P-loop and parts of the activation loop have been omitted for clarity. The hydrophobic spine residues (orange transparent surface) and the substrate-binding site (green transparent surface) are highlighted. (b) Prepared using PDB 1Y57, from which the simulation was initiated. (c) Prepared using a snapshot from a simulation, but the key features highlighted (for example, the positions of Trp260 and the Glu310-Arg409 salt bridge) are also seen in the crystal structure of inactive Src kinase (PDB 2SRC). The key hydrogen bond between protonated Asp404 and Asp386 is seen in other kinase crystal structures (for example, PDB 1JOW and 1M14).
Mentions: A conserved hydrophobic ‘spine’ in the protein kinase domain plays a critical role in maintaining the catalytically active conformation20. Consistent with this notion, the αC-out transition was accompanied in our simulations by the breaking of the hydrophobic spine (Leu325, Met314 and Phe405 of the DFG motif, and His384 in Src kinase) in the N-lobe (Figs 1c and 2b). Presumably, the protonation of Asp404 of the DFG motif leads the residue to disengage from Lys295; by the coupling of the DFG residues, this conformational change at Asp404 causes Phe405 to disengage from Met314, breaking the spine and repositioning the αC helix (Fig. 2b). We also observed that Trp260, the first residue of the kinase domain and a key residue in the cross-talk between the regulatory SH2/SH3 and kinase domains in Src2122, interacts with Leu325 and thus the hydrophobic spine (Fig. 2b). During the αC-out transition observed in the simulations, Trp260 changed position (Fig. 2b). Given that this repositioning of Trp260 is consistent with the different Trp260 conformations in active and inactive Src kinase structures (this conformation of Trp260 after the αC-out transition has been observed in X-ray structures of inactive Src kinase2324) and that it coincides with the αC-out transition in both of the two separate simulations in which the transition is observed (Fig. 1c, Plot A and Supplementary Fig. 2a, Plot A), we conclude that Trp260 is likely conformationally coupled with the hydrophobic spine, and the αC helix.

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