Limits...
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.


Related in: MedlinePlus

Structural and kinetic characteristics of FGFR1 complexes with the type I andtype II inhibitors PDA and ponatinib.(a) Chemical structures of PDA and ponatinib. (b) Active siteof FGFR1 kinase in complex with PDA (green carbons) as determined at2.09 Å resolution (Supplementary Table 5). The hinge region(yellow) and A-loop (orange) are highlighted.Fo−Fc OMIT electrondensity for PDA and the DFG motif is represented as a blue mesh contoured at3.0σ. Polar interactions are indicated as dotted lines.(c) Active site of FGFR1 kinase in complex with ponatinib (greycarbons) as determined at 2.33 Å resolution, withFo−Fc OMIT electrondensity for ponatinib and the DFG motif represented as a blue mesh contouredat 3.0σ. Colouring of FGFR1 as in b. (d)Kinetic value plot of association rate constant (kon)versus dissociation rate constant (koff). Rate constantswere determined using SPR at 298 K, pH=7.4. Theaffinities (KD) were calculated from the equationKD=koff/konand broken lines represent affinity isotherms. Data represent geometricmeans from at least three independent experiments; standard errors are shownas error bars (values and errors are presented in Supplementary Table 1). (e) Theassociation rate constant of ponatinib binding to FGFR1 as a function of pH,as measured by SPR at 298 K. The red line represents the resultof the non-linear fitting of the data to the 4 PL model(R2=0.968;pKa(Asp641)=6.25). (f) The associationrate constant of PDA binding to FGFR1 as a function of pH, as measured bySPR at 298 K.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4525181&req=5

f1: Structural and kinetic characteristics of FGFR1 complexes with the type I andtype II inhibitors PDA and ponatinib.(a) Chemical structures of PDA and ponatinib. (b) Active siteof FGFR1 kinase in complex with PDA (green carbons) as determined at2.09 Å resolution (Supplementary Table 5). The hinge region(yellow) and A-loop (orange) are highlighted.Fo−Fc OMIT electrondensity for PDA and the DFG motif is represented as a blue mesh contoured at3.0σ. Polar interactions are indicated as dotted lines.(c) Active site of FGFR1 kinase in complex with ponatinib (greycarbons) as determined at 2.33 Å resolution, withFo−Fc OMIT electrondensity for ponatinib and the DFG motif represented as a blue mesh contouredat 3.0σ. Colouring of FGFR1 as in b. (d)Kinetic value plot of association rate constant (kon)versus dissociation rate constant (koff). Rate constantswere determined using SPR at 298 K, pH=7.4. Theaffinities (KD) were calculated from the equationKD=koff/konand broken lines represent affinity isotherms. Data represent geometricmeans from at least three independent experiments; standard errors are shownas error bars (values and errors are presented in Supplementary Table 1). (e) Theassociation rate constant of ponatinib binding to FGFR1 as a function of pH,as measured by SPR at 298 K. The red line represents the resultof the non-linear fitting of the data to the 4 PL model(R2=0.968;pKa(Asp641)=6.25). (f) The associationrate constant of PDA binding to FGFR1 as a function of pH, as measured bySPR at 298 K.

Mentions: For our studies of the FGFR1 kinase domain, we have used a construct spanningresidues Ala458 to Glu765 of human FGFR1 that contains two mutations (Cys488Ala andCys584Ser) designed to stabilize the enzyme against covalent aggregation. Theprotein is non-phosphorylated after co-expression with PTP1B and purification fromEscherichia coli. An additional mutation of the catalytic aspartate(Asp623Ala) was introduced for NMR studies to increase further the yield of stableisotope-labelled protein. Our previous studies have shown that this mutation doesnot detectably perturb the structure of the FGFR1 kinase domain22. Inaddition, we have confirmed using surface plasmon resonance (SPR) that bindingparameters for a close analogue of the canonical FGFR1 inhibitor PD173074 (ref.23; henceforth referred to as PDA; Fig. 1a; Supplementary Fig.1) are unaltered for the Asp623Ala mutant relative to the kinase-activeform (data not shown).


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)

Structural and kinetic characteristics of FGFR1 complexes with the type I andtype II inhibitors PDA and ponatinib.(a) Chemical structures of PDA and ponatinib. (b) Active siteof FGFR1 kinase in complex with PDA (green carbons) as determined at2.09 Å resolution (Supplementary Table 5). The hinge region(yellow) and A-loop (orange) are highlighted.Fo−Fc OMIT electrondensity for PDA and the DFG motif is represented as a blue mesh contoured at3.0σ. Polar interactions are indicated as dotted lines.(c) Active site of FGFR1 kinase in complex with ponatinib (greycarbons) as determined at 2.33 Å resolution, withFo−Fc OMIT electrondensity for ponatinib and the DFG motif represented as a blue mesh contouredat 3.0σ. Colouring of FGFR1 as in b. (d)Kinetic value plot of association rate constant (kon)versus dissociation rate constant (koff). Rate constantswere determined using SPR at 298 K, pH=7.4. Theaffinities (KD) were calculated from the equationKD=koff/konand broken lines represent affinity isotherms. Data represent geometricmeans from at least three independent experiments; standard errors are shownas error bars (values and errors are presented in Supplementary Table 1). (e) Theassociation rate constant of ponatinib binding to FGFR1 as a function of pH,as measured by SPR at 298 K. The red line represents the resultof the non-linear fitting of the data to the 4 PL model(R2=0.968;pKa(Asp641)=6.25). (f) The associationrate constant of PDA binding to FGFR1 as a function of pH, as measured bySPR at 298 K.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Structural and kinetic characteristics of FGFR1 complexes with the type I andtype II inhibitors PDA and ponatinib.(a) Chemical structures of PDA and ponatinib. (b) Active siteof FGFR1 kinase in complex with PDA (green carbons) as determined at2.09 Å resolution (Supplementary Table 5). The hinge region(yellow) and A-loop (orange) are highlighted.Fo−Fc OMIT electrondensity for PDA and the DFG motif is represented as a blue mesh contoured at3.0σ. Polar interactions are indicated as dotted lines.(c) Active site of FGFR1 kinase in complex with ponatinib (greycarbons) as determined at 2.33 Å resolution, withFo−Fc OMIT electrondensity for ponatinib and the DFG motif represented as a blue mesh contouredat 3.0σ. Colouring of FGFR1 as in b. (d)Kinetic value plot of association rate constant (kon)versus dissociation rate constant (koff). Rate constantswere determined using SPR at 298 K, pH=7.4. Theaffinities (KD) were calculated from the equationKD=koff/konand broken lines represent affinity isotherms. Data represent geometricmeans from at least three independent experiments; standard errors are shownas error bars (values and errors are presented in Supplementary Table 1). (e) Theassociation rate constant of ponatinib binding to FGFR1 as a function of pH,as measured by SPR at 298 K. The red line represents the resultof the non-linear fitting of the data to the 4 PL model(R2=0.968;pKa(Asp641)=6.25). (f) The associationrate constant of PDA binding to FGFR1 as a function of pH, as measured bySPR at 298 K.
Mentions: For our studies of the FGFR1 kinase domain, we have used a construct spanningresidues Ala458 to Glu765 of human FGFR1 that contains two mutations (Cys488Ala andCys584Ser) designed to stabilize the enzyme against covalent aggregation. Theprotein is non-phosphorylated after co-expression with PTP1B and purification fromEscherichia coli. An additional mutation of the catalytic aspartate(Asp623Ala) was introduced for NMR studies to increase further the yield of stableisotope-labelled protein. Our previous studies have shown that this mutation doesnot detectably perturb the structure of the FGFR1 kinase domain22. Inaddition, we have confirmed using surface plasmon resonance (SPR) that bindingparameters for a close analogue of the canonical FGFR1 inhibitor PD173074 (ref.23; henceforth referred to as PDA; Fig. 1a; Supplementary Fig.1) are unaltered for the Asp623Ala mutant relative to the kinase-activeform (data not shown).

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.


Related in: MedlinePlus