Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp- Phe-Gly 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 H helix towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the C-4 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.
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