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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.


The role of the αC-β4 loop and molecular brake regions inthe DFG flip of FGFR1.(a) Comparison of αC-β4 loop and molecular brakeregions in FGFR1 and Abl kinase complexes with ponatinib. The structures ofthe FGFR1/ponatinib complex (PDB ID: 4V01) and the Abl/ponatinib complex(PDB ID: 3OXZ) are displayed in dark green and grey, respectively, with thebound ponatinib inhibitors displayed, respectively, in light green and grey.Relative to Abl, the αC helix of FGFR1 extends approximatelyone-half turn further, in part due to insertion of a Gly at position 539,and the ‘HxN hairpin' contains a Lys rather than a Proat the middle position. The molecular brake of FGFR1 is engaged via hydrogenbonds (dotted lines) from the side chain of Asn546 and likely inhibits theoutward motion of helix αC in FGFR1, whereas Abl, with a Gln atthe equivalent position and lacking the Gly insert at position 539, isunable to form the molecular brake interactions. (b) Schematicillustration of the interplay between the DFG flip, outward movement of theαC helix and the proposed role of the Asn546 molecular brakehydrogen bonds in FGFR1. The Asn546 hydrogen bonds (of which Abl lacks anequivalent) may need to be transiently disengaged (scissors) to facilitatethe αC-out, and hence DFG-out, movements. View in b as iffrom the left-hand side of a.
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f5: The role of the αC-β4 loop and molecular brake regions inthe DFG flip of FGFR1.(a) Comparison of αC-β4 loop and molecular brakeregions in FGFR1 and Abl kinase complexes with ponatinib. The structures ofthe FGFR1/ponatinib complex (PDB ID: 4V01) and the Abl/ponatinib complex(PDB ID: 3OXZ) are displayed in dark green and grey, respectively, with thebound ponatinib inhibitors displayed, respectively, in light green and grey.Relative to Abl, the αC helix of FGFR1 extends approximatelyone-half turn further, in part due to insertion of a Gly at position 539,and the ‘HxN hairpin' contains a Lys rather than a Proat the middle position. The molecular brake of FGFR1 is engaged via hydrogenbonds (dotted lines) from the side chain of Asn546 and likely inhibits theoutward motion of helix αC in FGFR1, whereas Abl, with a Gln atthe equivalent position and lacking the Gly insert at position 539, isunable to form the molecular brake interactions. (b) Schematicillustration of the interplay between the DFG flip, outward movement of theαC helix and the proposed role of the Asn546 molecular brakehydrogen bonds in FGFR1. The Asn546 hydrogen bonds (of which Abl lacks anequivalent) may need to be transiently disengaged (scissors) to facilitatethe αC-out, and hence DFG-out, movements. View in b as iffrom the left-hand side of a.

Mentions: Crystal structures and molecular dynamics simulations, using Abl kinase as a modelsystem, suggest that displacement of the αC helix away from the activesite facilitates the DFG flip in kinases, with the resulting‘αC-out' conformation being a potentialintermediate1136. The αC-β4 loop has beenproposed to act as an anchor for the αC helix to the catalytic core, andas a hinge for the αC helix during the transition from active to inactivestates of protein kinases3445. The substantial amide CSPs weobserved using NMR for residues in the αC-β4 loop in the DFG-outstate of FGFR1, coupled with significantly enhanced solvent exchange rates byHDX-MS, indicate that the transition from the active to the inactive state isaccompanied by a structural or dynamic perturbation. Compared with Abl, FGFR1contains an insert (Gly539) C-terminal to the αC helix and aconformationally significant substitution in the relatively conserved ‘HxNhairpin'3546 that follows (HPN in many kinasesincluding Abl; HKN in FGFRs). The HxN hairpin may function as a pivot for theoutward movement of the αC helix that is required to facilitate theexcursion of the DFG Phe side chain towards its ‘out'configuration46. The Gly539 insert results in extension of theC-terminal end of the αC helix of FGFR1 by around half a turn relative toAbl (Fig. 5a), and facilitates the formation of the molecularbrake hydrogen-bond network47 between the side chain of Asn546 andthe backbone atoms of His541 of the HxN motif. By contrast, Abl is unable to formthese hydrogen bonds to the HxN backbone. Asn546 is a key member of the triad thatforms the molecular brake in FGFR isoforms, and is the site of a number ofpathogenic gain-of-function mutations that are implicated in developmental disordersand cancers. The hydrogen–bond network involving Asn546 of FGFR1 would beexpected to stabilize the αC helix in its ‘in'orientation, thereby inhibiting the 'αC-out' movementrequired to effect the DFG flip (Fig. 5b). Thus, our analysissuggests that a distributed network of individual contributions from several regionsof the kinase structure conspires to hinder the DFG flip in FGFR1, and that the mostimportant of these is likely to reside in the αC-β4 loop region.This is interesting in light of a recent report that the N550K mutation in FGFR2(equivalent to Asn546 in FGFR1) confers resistance to the type I inhibitors PD173074and dovitinib, but not to ponatinib, which displays enhanced inhibitory potencyagainst this mutant relative to wild-type in BaF3 cell proliferation assays48. Our insights into the structural and dynamic influences on the DFGflip in FGFR1 corroborate the important role of the molecular brake in inhibitingbasal kinase activity in unphosphorylated FGFRs, and imply that its function (andits release by pathogenic mutations) may be intimately associated with its abilityto suppress the catalytically significant DFG flip11 by inhibitingthe outward movement of the αC helix.


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)

The role of the αC-β4 loop and molecular brake regions inthe DFG flip of FGFR1.(a) Comparison of αC-β4 loop and molecular brakeregions in FGFR1 and Abl kinase complexes with ponatinib. The structures ofthe FGFR1/ponatinib complex (PDB ID: 4V01) and the Abl/ponatinib complex(PDB ID: 3OXZ) are displayed in dark green and grey, respectively, with thebound ponatinib inhibitors displayed, respectively, in light green and grey.Relative to Abl, the αC helix of FGFR1 extends approximatelyone-half turn further, in part due to insertion of a Gly at position 539,and the ‘HxN hairpin' contains a Lys rather than a Proat the middle position. The molecular brake of FGFR1 is engaged via hydrogenbonds (dotted lines) from the side chain of Asn546 and likely inhibits theoutward motion of helix αC in FGFR1, whereas Abl, with a Gln atthe equivalent position and lacking the Gly insert at position 539, isunable to form the molecular brake interactions. (b) Schematicillustration of the interplay between the DFG flip, outward movement of theαC helix and the proposed role of the Asn546 molecular brakehydrogen bonds in FGFR1. The Asn546 hydrogen bonds (of which Abl lacks anequivalent) may need to be transiently disengaged (scissors) to facilitatethe αC-out, and hence DFG-out, movements. View in b as iffrom the left-hand side of a.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: The role of the αC-β4 loop and molecular brake regions inthe DFG flip of FGFR1.(a) Comparison of αC-β4 loop and molecular brakeregions in FGFR1 and Abl kinase complexes with ponatinib. The structures ofthe FGFR1/ponatinib complex (PDB ID: 4V01) and the Abl/ponatinib complex(PDB ID: 3OXZ) are displayed in dark green and grey, respectively, with thebound ponatinib inhibitors displayed, respectively, in light green and grey.Relative to Abl, the αC helix of FGFR1 extends approximatelyone-half turn further, in part due to insertion of a Gly at position 539,and the ‘HxN hairpin' contains a Lys rather than a Proat the middle position. The molecular brake of FGFR1 is engaged via hydrogenbonds (dotted lines) from the side chain of Asn546 and likely inhibits theoutward motion of helix αC in FGFR1, whereas Abl, with a Gln atthe equivalent position and lacking the Gly insert at position 539, isunable to form the molecular brake interactions. (b) Schematicillustration of the interplay between the DFG flip, outward movement of theαC helix and the proposed role of the Asn546 molecular brakehydrogen bonds in FGFR1. The Asn546 hydrogen bonds (of which Abl lacks anequivalent) may need to be transiently disengaged (scissors) to facilitatethe αC-out, and hence DFG-out, movements. View in b as iffrom the left-hand side of a.
Mentions: Crystal structures and molecular dynamics simulations, using Abl kinase as a modelsystem, suggest that displacement of the αC helix away from the activesite facilitates the DFG flip in kinases, with the resulting‘αC-out' conformation being a potentialintermediate1136. The αC-β4 loop has beenproposed to act as an anchor for the αC helix to the catalytic core, andas a hinge for the αC helix during the transition from active to inactivestates of protein kinases3445. The substantial amide CSPs weobserved using NMR for residues in the αC-β4 loop in the DFG-outstate of FGFR1, coupled with significantly enhanced solvent exchange rates byHDX-MS, indicate that the transition from the active to the inactive state isaccompanied by a structural or dynamic perturbation. Compared with Abl, FGFR1contains an insert (Gly539) C-terminal to the αC helix and aconformationally significant substitution in the relatively conserved ‘HxNhairpin'3546 that follows (HPN in many kinasesincluding Abl; HKN in FGFRs). The HxN hairpin may function as a pivot for theoutward movement of the αC helix that is required to facilitate theexcursion of the DFG Phe side chain towards its ‘out'configuration46. The Gly539 insert results in extension of theC-terminal end of the αC helix of FGFR1 by around half a turn relative toAbl (Fig. 5a), and facilitates the formation of the molecularbrake hydrogen-bond network47 between the side chain of Asn546 andthe backbone atoms of His541 of the HxN motif. By contrast, Abl is unable to formthese hydrogen bonds to the HxN backbone. Asn546 is a key member of the triad thatforms the molecular brake in FGFR isoforms, and is the site of a number ofpathogenic gain-of-function mutations that are implicated in developmental disordersand cancers. The hydrogen–bond network involving Asn546 of FGFR1 would beexpected to stabilize the αC helix in its ‘in'orientation, thereby inhibiting the 'αC-out' movementrequired to effect the DFG flip (Fig. 5b). Thus, our analysissuggests that a distributed network of individual contributions from several regionsof the kinase structure conspires to hinder the DFG flip in FGFR1, and that the mostimportant of these is likely to reside in the αC-β4 loop region.This is interesting in light of a recent report that the N550K mutation in FGFR2(equivalent to Asn546 in FGFR1) confers resistance to the type I inhibitors PD173074and dovitinib, but not to ponatinib, which displays enhanced inhibitory potencyagainst this mutant relative to wild-type in BaF3 cell proliferation assays48. Our insights into the structural and dynamic influences on the DFGflip in FGFR1 corroborate the important role of the molecular brake in inhibitingbasal kinase activity in unphosphorylated FGFRs, and imply that its function (andits release by pathogenic mutations) may be intimately associated with its abilityto suppress the catalytically significant DFG flip11 by inhibitingthe outward movement of the αC helix.

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.