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Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase.

Klein T, Vajpai N, Phillips JJ, Davies G, Holdgate GA, Phillips C, Tucker JA, Norman RA, Scott AD, Higazi DR, Lowe D, Thompson GS, Breeze AL - Nat Commun (2015)

Bottom Line: Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a 'DFG-out' state.Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib.We conclude that the αC-β4 loop and 'molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.

View Article: PubMed Central - PubMed

Affiliation: Discovery Sciences, AstraZeneca R&D, Alderley Park, Macclesfield SK10 4TG, UK.

ABSTRACT
Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp-Phe-Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called 'DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a 'DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-β4 loop and 'molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.

No MeSH data available.


Thermodynamic data for inhibitors binding to FGFR1 kinase domain.(a) Thermodynamic signatures for type I inhibitors binding to FGFR1derived by ITC at 298 K. Data shown are arithmeticmean±s.d. from at least two independent experiments (values anderrors are presented in SupplementaryTable 2). (b) van't Hoff plot visualization oftemperature-dependent FGFR1–ligand interactions measured by SPRfor PDA (blue circles), SU5402 (blue open squares) and ponatinib (redtriangles). (c) Thermodynamic reaction pathway models for FGFR1interacting with PDA (left) and ponatinib (right). The reaction coordinatedepicts the lowest energy continuous pathway between the free (centre of thefigure) and bound states (left for PDA complex; right for ponatinib complex)via the transition state, for free energy ΔG (green),enthalpy ΔH (blue) andentropy—TΔS (red).
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f2: Thermodynamic data for inhibitors binding to FGFR1 kinase domain.(a) Thermodynamic signatures for type I inhibitors binding to FGFR1derived by ITC at 298 K. Data shown are arithmeticmean±s.d. from at least two independent experiments (values anderrors are presented in SupplementaryTable 2). (b) van't Hoff plot visualization oftemperature-dependent FGFR1–ligand interactions measured by SPRfor PDA (blue circles), SU5402 (blue open squares) and ponatinib (redtriangles). (c) Thermodynamic reaction pathway models for FGFR1interacting with PDA (left) and ponatinib (right). The reaction coordinatedepicts the lowest energy continuous pathway between the free (centre of thefigure) and bound states (left for PDA complex; right for ponatinib complex)via the transition state, for free energy ΔG (green),enthalpy ΔH (blue) andentropy—TΔS (red).

Mentions: The apparently slow equilibrium between DFG-in and DFG-out conformations in FGFR1kinase suggests a high free-energy barrier for the DFG flip. We carried out adetailed analysis of the changes in enthalpy and entropy that accompany ligandbinding to enhance our understanding. For the selected type I inhibitors,isothermal titration calorimetry (ITC) experiments revealed exothermic bindingreactions (Fig. 2a, Supplementary Fig. 2, Supplementary Table 2), and the derived binding affinities werelargely in agreement with those determined by SPR. In contrast, for ponatinib,which binds to FGFR1 in a DFG-out conformation, the observed titration curve(Supplementary Fig. 2h) was ofpoor quality and did not allow derivation of thermodynamic parameters. As analternative to ITC, we analysed kinetic and equilibrium data from SPR as afunction of temperature, following the van't Hoff method, to provideindependent thermodynamic characterization of binding events. For ponatinib andtwo selected type I inhibitors (PDA and SU5402), the derived binding enthalpiesand entropies revealed another marked difference between the type II inhibitorponatinib and the selected type I inhibitors (Fig. 2b,Table 1). In the case of PDA and SU5402,van't Hoff analysis confirmed exothermic binding enthalpies (PDA,ΔH=−11.5 kcal mol−1;SU5402,ΔH=−14.2 kcal mol−1)and the data are in close agreement with the ΔH values of−12.1 kcal mol−1(PDA) and−12.4 kcal mol−1(SU5402) determined by ITC. Unexpectedly, the type II inhibitor ponatinib showedan endothermic ΔH value(ΔH=10.1 kcal mol−1)that indicates enthalpically unfavourable binding. Ponatinib and PDA exhibitcomparable van't Hoff free energies of binding (Table1) that are consistent with their very similar affinities measureddirectly by SPR; however, breaking this down into enthalpic and entropiccomponents revealed significant differences, as the binding of PDA and ponatinibwere determined to be enthalpy driven and entropy driven, respectively. Anendothermic enthalpy, as observed for the equilibrium between free andFGFR1-bound ponatinib, raises the possibility that the conformationalrearrangement required to effect the DFG flip in FGFR1 may also be associatedwith an enthalpic penalty (neglecting net contributions fromprotein–ligand and protein–solvent interactions ofponatinib). Furthermore, we established that the vascular endothelial growthfactor receptor (VEGFR) inhibitor tivozanib (AV-951) also binds to FGFR1 in aDFG-out mode (KD=1.3 μM by SPR)and does so endothermically by van't Hoff analysis (Supplementary Fig. 3), lending furthersupport to the notion that this may be a signature of a DFG-out binding mode forFGFR1, rather than a compound-specific characteristic of ponatinib.


Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase.

Klein T, Vajpai N, Phillips JJ, Davies G, Holdgate GA, Phillips C, Tucker JA, Norman RA, Scott AD, Higazi DR, Lowe D, Thompson GS, Breeze AL - Nat Commun (2015)

Thermodynamic data for inhibitors binding to FGFR1 kinase domain.(a) Thermodynamic signatures for type I inhibitors binding to FGFR1derived by ITC at 298 K. Data shown are arithmeticmean±s.d. from at least two independent experiments (values anderrors are presented in SupplementaryTable 2). (b) van't Hoff plot visualization oftemperature-dependent FGFR1–ligand interactions measured by SPRfor PDA (blue circles), SU5402 (blue open squares) and ponatinib (redtriangles). (c) Thermodynamic reaction pathway models for FGFR1interacting with PDA (left) and ponatinib (right). The reaction coordinatedepicts the lowest energy continuous pathway between the free (centre of thefigure) and bound states (left for PDA complex; right for ponatinib complex)via the transition state, for free energy ΔG (green),enthalpy ΔH (blue) andentropy—TΔS (red).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525181&req=5

f2: Thermodynamic data for inhibitors binding to FGFR1 kinase domain.(a) Thermodynamic signatures for type I inhibitors binding to FGFR1derived by ITC at 298 K. Data shown are arithmeticmean±s.d. from at least two independent experiments (values anderrors are presented in SupplementaryTable 2). (b) van't Hoff plot visualization oftemperature-dependent FGFR1–ligand interactions measured by SPRfor PDA (blue circles), SU5402 (blue open squares) and ponatinib (redtriangles). (c) Thermodynamic reaction pathway models for FGFR1interacting with PDA (left) and ponatinib (right). The reaction coordinatedepicts the lowest energy continuous pathway between the free (centre of thefigure) and bound states (left for PDA complex; right for ponatinib complex)via the transition state, for free energy ΔG (green),enthalpy ΔH (blue) andentropy—TΔS (red).
Mentions: The apparently slow equilibrium between DFG-in and DFG-out conformations in FGFR1kinase suggests a high free-energy barrier for the DFG flip. We carried out adetailed analysis of the changes in enthalpy and entropy that accompany ligandbinding to enhance our understanding. For the selected type I inhibitors,isothermal titration calorimetry (ITC) experiments revealed exothermic bindingreactions (Fig. 2a, Supplementary Fig. 2, Supplementary Table 2), and the derived binding affinities werelargely in agreement with those determined by SPR. In contrast, for ponatinib,which binds to FGFR1 in a DFG-out conformation, the observed titration curve(Supplementary Fig. 2h) was ofpoor quality and did not allow derivation of thermodynamic parameters. As analternative to ITC, we analysed kinetic and equilibrium data from SPR as afunction of temperature, following the van't Hoff method, to provideindependent thermodynamic characterization of binding events. For ponatinib andtwo selected type I inhibitors (PDA and SU5402), the derived binding enthalpiesand entropies revealed another marked difference between the type II inhibitorponatinib and the selected type I inhibitors (Fig. 2b,Table 1). In the case of PDA and SU5402,van't Hoff analysis confirmed exothermic binding enthalpies (PDA,ΔH=−11.5 kcal mol−1;SU5402,ΔH=−14.2 kcal mol−1)and the data are in close agreement with the ΔH values of−12.1 kcal mol−1(PDA) and−12.4 kcal mol−1(SU5402) determined by ITC. Unexpectedly, the type II inhibitor ponatinib showedan endothermic ΔH value(ΔH=10.1 kcal mol−1)that indicates enthalpically unfavourable binding. Ponatinib and PDA exhibitcomparable van't Hoff free energies of binding (Table1) that are consistent with their very similar affinities measureddirectly by SPR; however, breaking this down into enthalpic and entropiccomponents revealed significant differences, as the binding of PDA and ponatinibwere determined to be enthalpy driven and entropy driven, respectively. Anendothermic enthalpy, as observed for the equilibrium between free andFGFR1-bound ponatinib, raises the possibility that the conformationalrearrangement required to effect the DFG flip in FGFR1 may also be associatedwith an enthalpic penalty (neglecting net contributions fromprotein–ligand and protein–solvent interactions ofponatinib). Furthermore, we established that the vascular endothelial growthfactor receptor (VEGFR) inhibitor tivozanib (AV-951) also binds to FGFR1 in aDFG-out mode (KD=1.3 μM by SPR)and does so endothermically by van't Hoff analysis (Supplementary Fig. 3), lending furthersupport to the notion that this may be a signature of a DFG-out binding mode forFGFR1, rather than a compound-specific characteristic of ponatinib.

Bottom Line: Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a 'DFG-out' state.Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib.We conclude that the αC-β4 loop and 'molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.

View Article: PubMed Central - PubMed

Affiliation: Discovery Sciences, AstraZeneca R&D, Alderley Park, Macclesfield SK10 4TG, UK.

ABSTRACT
Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp-Phe-Gly (DFG) motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so-called 'DFG flip'. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a 'DFG-out' state. Here we use conformationally selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the αH helix, towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the αC-β4 loop and 'molecular brake' regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.

No MeSH data available.