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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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Concerted conformational change simulated in Src kinase up on protonation.(a) The movement of the αC helix in the MD simulations. Starting from the active conformation (Simulation—Start), the αC helix (cyan transparent) rotated outwards by about 120° (cyan solid) leading to a salt bridge between Glu310 and Arg409 (Simulation—End). The structure at the end of the simulation resembles that in the crystal structure (red) of autoinhibited Src kinase (Experiment—Inactive, PDB entry 2SRC). (b) The location of the contiguous network of residues involved in the concerted conformational change. Green letters denote the approximate location of conformational changes quantified in c; a more detailed view of the key residues involved in the conformational change is shown in Fig. 2b,c. (c) The structural parameters (contact area, salt bridge or hydrogen bond distance and r.m.s.d.) characterizing the conformational change shown as functions of simulation time. Light blue is used to highlight the narrow time window in which the concerted conformational change occurred.
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f1: Concerted conformational change simulated in Src kinase up on protonation.(a) The movement of the αC helix in the MD simulations. Starting from the active conformation (Simulation—Start), the αC helix (cyan transparent) rotated outwards by about 120° (cyan solid) leading to a salt bridge between Glu310 and Arg409 (Simulation—End). The structure at the end of the simulation resembles that in the crystal structure (red) of autoinhibited Src kinase (Experiment—Inactive, PDB entry 2SRC). (b) The location of the contiguous network of residues involved in the concerted conformational change. Green letters denote the approximate location of conformational changes quantified in c; a more detailed view of the key residues involved in the conformational change is shown in Fig. 2b,c. (c) The structural parameters (contact area, salt bridge or hydrogen bond distance and r.m.s.d.) characterizing the conformational change shown as functions of simulation time. Light blue is used to highlight the narrow time window in which the concerted conformational change occurred.

Mentions: Protonation of the DFG aspartate, however, enabled the transition to the αC-out conformation: in four out of the eight separate 100-ns-timescale simulations (the eight simulations amounted to a total of 2,400 ns), the kinase departed from the active αC-in conformation, as indicated by the breaking of the Lys290-Glu310 salt bridge (Supplementary Fig. 1a). The protonation produces a conformational change of Asp404 in the DFG motif (with which Phe405 of the motif is coupled) and, in turn, induces the αC-out transition. Without using any crystal structure information about the αC-out conformation, the simulations produced the correct αC-out conformation, in which the helix is ~2 Å backbone root mean squared deviation (r.m.s.d.) from the inactive conformation as captured in PDB entry 2SRC (Fig. 1c, Plot N and Supplementary Fig. 2a, Plot N), and ~8 Å backbone r.m.s.d. away from the initial (active) αC-in conformation.


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

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

Concerted conformational change simulated in Src kinase up on protonation.(a) The movement of the αC helix in the MD simulations. Starting from the active conformation (Simulation—Start), the αC helix (cyan transparent) rotated outwards by about 120° (cyan solid) leading to a salt bridge between Glu310 and Arg409 (Simulation—End). The structure at the end of the simulation resembles that in the crystal structure (red) of autoinhibited Src kinase (Experiment—Inactive, PDB entry 2SRC). (b) The location of the contiguous network of residues involved in the concerted conformational change. Green letters denote the approximate location of conformational changes quantified in c; a more detailed view of the key residues involved in the conformational change is shown in Fig. 2b,c. (c) The structural parameters (contact area, salt bridge or hydrogen bond distance and r.m.s.d.) characterizing the conformational change shown as functions of simulation time. Light blue is used to highlight the narrow time window in which the concerted conformational change occurred.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Concerted conformational change simulated in Src kinase up on protonation.(a) The movement of the αC helix in the MD simulations. Starting from the active conformation (Simulation—Start), the αC helix (cyan transparent) rotated outwards by about 120° (cyan solid) leading to a salt bridge between Glu310 and Arg409 (Simulation—End). The structure at the end of the simulation resembles that in the crystal structure (red) of autoinhibited Src kinase (Experiment—Inactive, PDB entry 2SRC). (b) The location of the contiguous network of residues involved in the concerted conformational change. Green letters denote the approximate location of conformational changes quantified in c; a more detailed view of the key residues involved in the conformational change is shown in Fig. 2b,c. (c) The structural parameters (contact area, salt bridge or hydrogen bond distance and r.m.s.d.) characterizing the conformational change shown as functions of simulation time. Light blue is used to highlight the narrow time window in which the concerted conformational change occurred.
Mentions: Protonation of the DFG aspartate, however, enabled the transition to the αC-out conformation: in four out of the eight separate 100-ns-timescale simulations (the eight simulations amounted to a total of 2,400 ns), the kinase departed from the active αC-in conformation, as indicated by the breaking of the Lys290-Glu310 salt bridge (Supplementary Fig. 1a). The protonation produces a conformational change of Asp404 in the DFG motif (with which Phe405 of the motif is coupled) and, in turn, induces the αC-out transition. Without using any crystal structure information about the αC-out conformation, the simulations produced the correct αC-out conformation, in which the helix is ~2 Å backbone root mean squared deviation (r.m.s.d.) from the inactive conformation as captured in PDB entry 2SRC (Fig. 1c, Plot N and Supplementary Fig. 2a, Plot N), and ~8 Å backbone r.m.s.d. away from the initial (active) αC-in conformation.

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